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The ultrastructure of primary cilia in quiescent 3T3 cells
CopyrIght 0 1980 by Academic Prera. Inc. ,411 righta of reproductmn in any form revswed M)l4-4X27/80/040427-I ISO2.00/0
Experimental
Cell Research 126 (1980) 427-437
THE ULTRASTRUCTURE
OF PRIMARY
IN QUIESCENT GUENTER
3T3 CELLS
ALBRECHT-BUEHLER
Cold Spring Harbor
Laboratory,
CILIA
and ANNE BUSHNELL
Cold Spring Harbour,
NY 11724, USA
SUMMARY Electron microscopy was used to investigate primary cilia in quiescent 3T3 cells. As in the case of primary cilia of other cell types, their basal centriole was found to be a focal point of numerous cytoplasmic microtubules which terminate at the basal feet. There are also intermediate filaments which appear to converge at the basal centriole. Cross-striated fibers of microtubular diameter, reminiscent of striated rootlets of ordinary cilia, appear associated with the proximal end of the basal centriole. Usually less than nine cross-banded basal feet surround the basal centriole,in a well-defined plane perpendicular to the centriolar axis. The ciliary shaft was found to be enttrely enclosed in the cytoplasm of fully flattened cells. In rounded cells, it could be found extending to the outside of the cell. Periodic striations along the entire shaft were observed after preparing the cells in a special way. The tip of the shaft showed an electron-dense specialization. Several unusual forms of primary cilia were observed which were reminiscent of olfactory flagella or retinal rods. Using tubulin antibody for indirect immunofluorescence, a fluorescent rod is visible in the cells [18] which we demonstrate is identical with the primary cilium.
Primary (rudimentary, solitary) cilia and their biological function are still an enigmatic and often unknown aspect of animal cell physiology. They are defined as single cilia which grow out of one of the centrioles during interphase in otherwise unciliated animal cells. Before mitosis, the cilium shaft is disassembled [21] and only unciliated centrioles are found at the spindle poles [4, 291. Many of the primary cilia remain, for the most part, inside the cell body. In contrast to normal, motile cilia with the well-known 9+2 pattern, primary cilia show a 9+0 pattern [5]. Although discovered in 1898 [35], primary cilia were first carefully investigated in 1961 and 1962 by Barnes [5] and Sorokin [25], who described them in mouse hypophysis as well as in fibroblasts and smooth muscle cells of neonatal chicken. By the year 1967 Scherft
& Daems [24] listed 36 avian and mammalian tissues which contain primary cilia. In 1971 Wheatley [33, 341 described .such cilia in cultured cell lines such as BHK21 and 3T6. It seems, therefore, that primary cilia are quite common centriolar specializations in vivo and in vitro. According to the work of Sorokin [25] and Wheatley [33, 341 primary cilia form after a vacuole (ciliary vacuole) has attached to the distal end of one of the c,entrioles. Subsequently, the ciliary shaft elongates into the, vacuole. The shaft has a membrane around it which appears to be derived from the basal part of the ciliary vacuole. In many cases, the distal part of the elongated vacuole connects to the outside of the cell, thus leaving the now partially. external cilium located in a more or less deep basal cup. The connection Exp Cell Res 126 (19801
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Albrecht-Buehler and Bushnell
of the ciliary vacuole to the outside does not always occur, especially in cultured cells. The biological function of primary cilia is unknown. Since retinal rods [8], olfactory flagellae [20] and kinocilia may be considered specializations of primary cilia, even though only retinal rods show a 9+0 pattern, one may suspect them of having a sensory function l-51, perhaps that of a chemoreceptor [16]. It was also noted that ciliation of centrioles correlated with a reduction of mitotic activity. Furthermore, it was found that blocking mitosis with colcemid induced primary cilia in CHO cells [7]. These findings led to the suggestion that primary cilia may be involved in control of the cell cycle [4, 9, 311, although this interpretation has been challenged [ 121. Furthermore, it was recently shown that PtKl cells resorb their primary cilium as late as during early spindle formation [21], which makes it less likely that primary cilia control cell division and more likely that they are subject to this control. Aside from these possibilities, using indirect immunofluorescence we recently found [l] that the primary cilia in migrating 3T3 cells was oriented parallel to the substrate and predominantly pointing in the direction of migration. In addition, we found in migrating 3T3 cells one centriole oriented preferentially parallel, the other perpendicular to the substrate [2]. The parallel centriole could be ciliated and, thus, easily observable by immunofluorescence. It seems that these observations add an aspect of spatial directionality to the suspected sensory function of primary cilia. In view of the difficulties of interpreting the biological function of primary cilia, it may seem appropriate to collect more information about them. Therefore, we studied the ultrastructure of primary cilia in quiescent 3T3 cells which express primary Exp Cell Res 126 (1980)
cilia at a frequency of 100% [l]. Several of our findings confirm previous descriptions of primary cilia in other cells. We include such features of the primary cilia of quiescent 3T3 cells in this report, nevertheless, because they may help to establish the common features of primary cilia.
METHODS Cell cultures. Swiss 3T3 cells, a kind gift of Dr Howard Green, Massachusetts Institute of Technology, were grown in Dulbecco’s modified Eagle’s medium (Grand Island Biological Co., Grand Island, N.Y .) supplemented with 10% calf serum (Microbial. Assoc., Bethesda, Md) in a 10% CO, atmosphere with saturated humidity. After reaching confluency at day 3, the cells were left in culture (without medium change) for another 7-9 days, in order to insure quiescence. Subsequently, they were processed for electron microscopy Immunojluorescence. Following a 30 min fixation of the cells in 3.5% formaldehyde in phosphate buffered saline (PBS), the cells were acetone extracted and incubated for 30 min at 37°C with rabbit antibovine brain tubulin antibody, a kind gift of Dr Frank Solomon, Massachusetts Institute of Technology.
Electron microscopy Preparation method I. Cells in dishes were fixed for 30 min with 2 % glutaraldehyde in 0.1 M phosphate buffer (PB) at DH 7.2-7.4. After nest-fixation for 30 min in 1% 0~6, in PB the cells were dehydrated in a graded ethanol-water series. Subsequently, the cells were left overnight under a layer of Epon 812 (Ted Pella Corp., Tuston, Calif.), reembedded in Epon 812, which was polymerized overnight at 70°C. The polymerized resin was popped out the dish, cut, trimmed, and sectioned with a diamond knife (DuPont Instruments, Wilmington, Del.) on an LKB ultrotome III. Specimens were examined with a Philips 201 electron microscope. Preparation method II. Same as I, except that cells were fixed in 2.5% glutaraldehyde in 0.1 M PB containing 0.1 M sucrose. Furthermore, before the cells were overlayed with Epon, they were left overnight in a 1 : 1 ethanol-Epon mixture.
RESULTS Centrioles and primary cilia in 3T3 cells Fig. 1 compares the ultrastructure of centrioles with that of primary cilia in 3T3 cells. Fig. 1b shows a typical longitudinal section through a centriole demonstrating the taper-
Primary cilia in 3T3 cells
429
Fig.
2. Comparison of normal centrioles (b-j’) with primary cilia of 3T3 cells (a, g-k) in longitudinal and cross-section. Bipointed arrows indicate approhimate location of the cross-sections. sl, Small central lumen;
ol, open lumen; bf basal feet; WC, ciliary vacuole. Arrowhead in h points to one of the alar sheets; arrowheads in e point to the electron-dense plaques which line 3 consecutive triplet blades. Bar, 0.5 pm.
ing of the wall microtubules toward the distal end. Two-thirds of the lumen of the centriole is filled with fibrous material beginning at the distal end. In axial sections (fig. lc-J) the fibrous material leaves a small central lumen (fig. 1d, e, “4”) which extends down the centriolar axis as far as the fibrous material. Near the proximal end the entire centriolar lumen appears empty. In 19 serial sections through centrioles in 3T3 cells we could not observe a cartwheel at the proximal end. Quite frequently electron opaque plaques appeared associated with the outer side of two or three consecutive blades near the proximal end (fig. 1e, arrowheads). In longitudinal section, the basal centriole of a primary cilium appears quite similar to a centriole (fig. la). However, basal feet which are not observed in unciliated centrioles (fig. 1a, “bf ‘) and which
are the focal point of numerous microtubules appear connected to the wall. They will be described in more detail below. In fig. 1a, we used preparation method II, for better visibility of the ciliary vacuole (fig. la, g, “vat”). Near the bottom the membrane of the ciliary vacuole appears quite dense (see also the other figures of this paper). A periodic striation extending up the shaft can be observed (see also below) indicating the presence of a ciliary necklace as in normal cilia. In axial section (fig. 1g-k) the basal centriole appears quite similar to the normal centriole. However, in addition to the basal feet (fig. 1i, “bf’), transitional fibers or alar sheets (fig. lh, arrowhead) [3] can be observed, as in ordinary cilia. Above the distal end, the section through the ciliary shaft (fig. 1g) shows the usual microtubular doublet array in the 9+0 pattern. The ciliary membrane and the Exp Cell Rrs 126 (1980)
Fig. 2. Identification of the primary cilium in immunofluorescence using tubulin antibody. (The long dark line in c is a fold in the section.) The panels show the same 3T3 cell in phase-contrast (a); indirect immunofluorescence (b; arrow points to the fluorescent rod); transmission electron microscopy (ventral, c;
higher up, d; arrow points to primary cilium in identical position and orientation as the fluorescent rod). e, Primary cilium at higher magnification. All microtubules are destroyed due to the preparation for immunofluorescence. Bar, (a, d) 20 pm; (e) 1 pm.
membrane of the ciliary vacuole in crosssection are usually separated.
[18] to reveal a rod-like fluorescent structure which appeared to be a nucleation center of cytoplasmic microtubules. Osborn & Weber, as well as subsequent investigators [l, 311, interpreted this rod as the primary cilium. The formal proof for this identification has not yet been given. Fig. 2
Primary cilia in indirect immunofluorescence Indirect immunofluorescence using tubulin antibody was shown by Osborn & Weber Exp
Cell
RPS 126 (/9X0)
Primary cilia in 3T3 cells
431
Fig. 4. Details of the basal feet structure. (u-d) Grazing sections through the tip of basal feet demonstrating their planar arrangement in a quite reproducible position. Arrows point to portions of the sectioned basal feet; (e-i) cross-banding of single basal feet (arrowheads). (g) One of the observed basal centrioles with two basal feet (arrows) above each other. Bar, 0.5 pm.
Fig. 3. Primary cilium of a post-telophase 3T3 cell extending to the outside of the cell. Bar, 1 pm.
attempts to close this gap by comparing the fluorescence micrograph and the electron micrograph of the same 3T3 cell. Beside the phase micrograph of a 3T3 cell (fig. 2a) fig. 2b shows the fluorescent micrograph
of the same cell with the fluorescent rod. Before this micrograph was taken, the cell had been formaldehyde-fixed, acetoneextracted, and fluorescence-stained. Subsequently, the cell was post-fixed with glutaraldehyde and osmium tetroxide and processed for electron microscopy. Fig. 2c shows a ventral section parallel to the substrate. The nucleoli pattern, general shape of cell and nucleus demonstrate the identity between this cell and the one photographed in phase contrast. In a higher section (fig. 2 d) the primary cilium appears in the identical location and orientation as the fluorescent rod. At higher magnification (fig. 2e) it is apparent that the formaldehyde-acetone treatment destroyed all microtubules. Nevertheless, basal centriole, ciliary vacuole, shaft and necklace of the structure can clearly be distinguished. Electron microscopy
of primary cilia
The following description is based on approx. 600 electron micrographs of primary cilia in quiescent 3T3 cells which were Exp CellRes 126 (1980)
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Albrecht-Buehler and Bushnell
Fig. 5. Grazing section through two basal feet of a primary cilium. The shaft (recognizable by the crossstriations) extends in the direction of the arrow. The
section illustrates the elaborate radial organization of cytoplasmic microtubules (mt), but also of intermediate filaments (iJ) around primary cilia. Bar, 1 ,um.
sectioned parallel to the substrate. With only a few exceptions, it extended parallel to the substrate. In all flattened cells we found the cilium to be entirely inside the cytoplasm. Serial sections as well as numerous single sections showed the primary cilium surrounded from all sides by cytoplasm (see, e.g. fig. 9). In still rounded, reflattening, post-telophase cells, however, we found several cases of cilia which extended to the outside of the cell (fig. 3). Most tissue cells in vivo have a more rounded shape than flattened cultured cells. Since their primary cilia in most cases were found to emerge to the outside [4, 12, 24,
251, it seems possible that the complete enclosure of primary cilia in 3T3 cells is caused by their flattening on the artificial substrate. The basal centriole of primary cilia was found to have a diameter of 0.26 pm and lengths between 0.41 and 0.51 pm. In 27 sections which showed the entire ciliary shaft we found a shaft length of 4 pm (S.D. 1.5 pm). However, there were cilia as long as 7 pm. The primary cilium was usually found near a nuclear indentation. The distance between the basal centriole and the nuclear envelope was measured in 130 micrographs which showed the cilium to-
Exl, Cell Res 126(1980)
Primary cilia in 3T3 cells
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Fig. 6. Striated “rootlets” (arrows) of about 250 A diameter connected to basal centrioles. Bar, 0.5 pm.
gether with the nucleus and yielded a surprisingly constant distance of 1.2 pm (S.D. 0.5 pm). A constant distance between basal centriole and nuclear envelope seems to support the suggestion of a material connection between them [6, 171. We were unable to find an obvious connection in the micrographs. However, it may be possible to observe it only after extraction of the cytoplasmic matrix as in the quoted experiments. The two centrioles of a pair are usually oriented at right angles to each other. In sections of quiescent 3T3 cells which showed the second unciliated centriole, we rarely found this to be the case. Most of the time, the associated centriole was at some oblique angle relative to the axis of the basal centriole of a primary cilium. Basal feet
The most striking ultrastructural feature of primary cilia is the convergence of cytoplasmic microtubules [30] toward the basal feet [15]. The focussing of cytoplasmic microtubules towards primary cilia has also
Fig. 7. primary primary also fig.
Dense tip specializations of three different cilia (cf also fig. 3). Arrows in c show two cilia enclosed in the same ciliary vacuole (cf 9e). Bar, 0.5 pm.
been demonstrated by indirect immunofluorescence [ 181.The basal feet are located within a plane collar around the centriolar wall. This is apparent in axial sections through this collar or even better, in grazing longitudinal sections which cut through the tips of the basal feet such as shown in fig. 4a-d. In three exceptional cases, however, we found two basal feet, one above the other, in the axial direction (fig. 4g, arrowheads). Fig. 4 also illustrates that the collar of basal feet is located at a rather reproducible distance from the proximal end. Measurements of 57 sections showed this distance to be 0.3 pm (S.D. 0.1 pm). Confirming earlier observations [ 10, 281, we found that basal feet have a conical shape with a triple cross-banding (fig. 4e-i, arrowheads). It may also be noteworthy that in three perfectly axial cross-sections through the collar of basal feet we found less than 9 basal feet surrounding the centriolar wall (see also fig. 1i). Exp Cell Res 126 (1980)
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and Bushnell
around the basal centriole yields an estimate of 130 microtubules which converge to one basal centriole. Assuming that the second centriole of a pair is surrounded by equally many microtubules, one would estimate that 260 microtubules converge to the centriole pair. “Rootlets”
Striated fibers were regularly observed near primary cilia. Most of them appeared to arise from the proximal end of the basal centriole. Some cases showed quite elaborate arrays of these fibers (fig. 6a) or even a large loop (fig. 6b). The visibility of the striated fibers was hampered by the surrounding cytoplasmic matrix which made it impossible to follow them through serial sections. Therefore, we do not know where they terminate in the cell. They showed a diameter of 250 A and a periodicity of 730 A (S.D. 20 A) and appeared significantly difFig. 8. Periodic shaft striations observed in primary ferent from the striated rootlets in ordinary cilia using fixation method II. Bar, 0.5 pm. cilia [3, 271, which have a periodicity of 650 A and a diameter of 700-800 A. Since The basal feet are the focal point of an we never observed any other kind of strielaborate array of cytoplasmic microtu- ated “rootlets”, it is not clear whether these bules (fig. 5). As illustrated in fig. 6, there structures are rootlets in the sense of ordiare also numerous intermediate filaments nary cilia. The diameter of 250 8, of the which appear to run parallel to microtu- striated rootlets suggests the possibility that bules towards the basal centriole. In addi- they are special microtubules which are tion to these fibrous structures, numerous periodically labelled with associated comvesicles were observed surrounding the ponents. cilia. Occasionally, we found the previously described [13] virus-like particles near the The ciliary shaft basal centrioles. At the base of the ciliary shaft the expected It is apparent from fig. 5 that the basal 9+0 doublet pattern is quite regular. Higher centriole is a major organization center for up in the ciliary shaft the 9 microtubular cytoskeletal fibers. This micrograph shows doublets appear to become disorganized. about 35 microtubules converging to the Occasionally we saw material connections basal centriole. With a section thickness of between the ciliary shaft and the surroundabout 1000 A one can estimate the average ing ciliary vacuole and even unidentified spatial sector occupied by one microtubule. inclusions. The latter were only seen in Extrapolating this value to an entire sphere highly vacuolarized cells suggesting the Exp CellRes 126(1980)
Primary cilia in 3T3 cells
435
Fig. 9. Unusual forms of primary cilia. (a, b) Bulging shafts with dense inclusions; (c) enclosed large vacuole; (d) bulging dense tip; (e) two parallel cilia
in the same ciliary vacuole; Bar indicates 0.5 pm. (f) unusually long shaft. Bar, (a-e) 0.5 pm; (f) 1 pm.
possibility that these were the result of a degeneration process. Whenever the sections contained the tip of the ciliary shaft, we observed electrondense material concentrated in the tip, demonstrating that the organization of the ciliary shaft is not uniform along its length. Fig. 7 shows three different examples which illustrate how reproducible this tip specialization is. A similar tip was shown in fig. 3. The tip density could become quite expanded with included vacuoles and even dense substructures. Fig. 9d shows such a bulging tip which is quite similar to a micrograph of Sorokin’s [25] obtained from a fibroblast in organ culture. Tip densities are not observed in ordinary cilia of vertebrates
primary cilia after cells recovered from colcemid treatment. We found 27 cases of similar striations extending along the entire ciliary shaft after the cells had been treated by preparation method II (fig. 8). Using the normal preparation I, we found such extended striations in only two cases, although a short stretch of striations is generally observed at the base of the ciliary shaft. In the literature, these short stretches have been termed the ciliary necklace. Preparation method II, which obviously destroys all membranes, including that of the ciliary vacuole and shaft, may facilitate the observation of the ciliary necklace extending all the way up the ciliary shaft. Measuring the periodicity of the striations in 17 cilia over at least 8 periods each we found a periodicity of 430 8, (SD. 30 A). In ordinary cilia the dynein repeat is 143 A and the spokes repeat with a periodicity of 860 8, every three unequally spaced spokes
[231. Shaft striations In several cases, Archer & Wheatley [4] observed a conspicuous cross-striation of
EXP Cell Res 126 (/MO)
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Albrecht-Buehler
and Bushnell
[32]. None of these periodicities coincides with the periodicity of the shaft striations. Therefore, it seems unlikely that these striations are related to the spokes or dynein organization of ordinary cilia. It should be noted, though, that three dynein repeats would generate 430 A. It is also noteworthy that two periods of 430 A would generate the 860 8, of the spokes repeat. Interestingly, the outer rod segments of retinal rods are spaced by 400 A [ 191,which is the same distance as the observed shaft striations. Unusual structures of primary
cilia
Several primary cilia showed quite unusual ultrastructures. Some of them are shown in fig. 9. Unusually long ciliary shafts (fig. 9f, and the enlarged dense tip shown in fig, 9d have been mentioned above. Confirming other observations on different cells [5, 341 we found several cases with two parallel cilia enclosed in the same ciliary vacuole (figs 7 c , 9 e). Furthermore, we found several cases with dramatically enlarged and bulging ciliary shafts (fig. 9a, b) including cases with large enclosed vacuoles (fig. SC). More frequently, small vacuoles were observed inside the shaft as well as dense bodies such as seen in the lefthand side of fig. 9a. Such cases were observed using normal preparation method I as well as method II. Nevertheless, the possibility cannot be excluded that preparation artifacts or degeneration processes in the quiescent cells produced these unusual forms. We mention their existence for the sake of completeness. DISCUSSION The present study describes primary cilia in quiescent 3T3 cells as elaborately structured organelles quite similar to primary E.l-l, Cell Res 126 (1980)
cilia in other cell types. Their basal centrioles are a focal point of cytoplasmic microtubules, but apparently also of intermediate fdaments, and of striated fibers of microtubular diameter, which may be related to the striated rootlets in ordinary cilia. There have been earlier suggestions of an association between intermediate filaments and centrioles. The caps of intermediate filaments in spreading BHK21 cells contain centrioles [26]. A rather striking association between centrioles and intermediate filaments has been observed in duodenal epithelium [22]. In this case a tight ring of such filaments encircles the centriole in a plane perpendicular to its axis. It appears that basal centrioles of primary cilia bring the converging cytoplasmic microtubules closer to the centriolar wall than normal centrioles. Although normal centrioles as seen in electron microscopy of whole cells are more or less radially surrounded by cytoplasmic microtubules, the microtubules rarely seem to come closer than 0.5-l pm to the centriolar wall. In contrast, isolated centrioles can nucleate microtubules at the wall [13, 141 which is surrounded by the so-called pericentriolar material. Telzer & Rosenbaum, using centrioles of HeLa cells, have recently shown that the microtubule nucleating capacity of isolated centrioles is most pronounced in centrioles from mitotic cells [29]. Considering the rather spectacular microtubular organization around basal centrioles of primary cilia in quiescent 3T3 cells, one may suggest that GO presents another phase of the cell cycle where centriolar nucleation of cytoplasmic microtubules is enhanced. The observation that less than nine basal feet can be found around the wall of the basal centriole, but also the observation of 2-3 electron-opaque plaques on consecu-
Primary cilia in 3T3 cells tive blades in centrioles, suggest that the nine blades of a centriole are not equivalent. It is tempting to speculate that such asymmetries in the otherwise so perfectly symmetrical centrioles are important for the postulated role of centrioles in controlling cell migration or other directional functions of cells. The cross-striations along the ciliary shaft which appear to be an extension of the ciliary necklace may indicate that some, if not all, primary cilia amplify a biological function of the ciliary necklace. It appears that the ciliary necklace binds calcium [ 111. P. Satir has suggested (personal communication) that the shaft of the primary cilium may serve as a calcium source or sink. The ciliary shaft is isolated from the rest of the cytoplasma either by the ciliary vacuole or because it extends to the outside. In either case, it appears to be accessible only through the basal centriole. Consequently, one may contemplate a directional release of calcium from the shaft into the cell with all its ramifications as to calcium-sensitive cellular and molecular processes which it may render directional. It remains to be seen whether the shaft of the primary cilium binds calcium in a similar way as ordinary cilia do. We are grateful for Dr James D. Watson’s support. The photographic work of Mr Ted Lukralle and the skillful typing of MS Madeline Szadkowski are gratefully acknowledged. The work was supported by the Cold Spring Harbor Laboratory Cancer Center grant from the NCI.
437
3. Anderson, R G W, J cell biol54 (1972) 246. 4. Archer, F L & Wheatley, D N, J anat 109 (1971) ^-LII. 5. Barnes, B G, J ultrastruct res 5 (1961) 453. 6. Bornens, M, Nature 270 (1977) 80. 7. Brinkley, B R & Stubblefield, E, J ultrastruct res 19 (1967) 1. 8. DeRobertis, E, J biophys biochem cytol 2 (1956) 319. 9. Dingemans, K P, J cell bio143 (1969) 361. 10. Doolin, P F & Birge, W J, J cell biol29 (1966) 333. II. Fisher, G, Kaneshiro, E S & Peters, P D, J cell biol69 (1976) 429. 12. Fonte, V G, Searls, R L & Hilfer, S R, J cell biol 49 (1971) 226. 13. Gould, R R & Borisy, G G, J cell bio173 (1977)601. 14. Heidemann. S R. Sanders. G & Kirschner. M W. Cell 10 (1977) 337. 15. Kalnins, V I & Porter, K R, Z Zellforsch 100 (1963) 1. 16. Muneer. B L. Am i anat 103 (1958) 275. 17. Nadizhdina,‘E S, “Fais, D & ‘Chentsov, Y S, Eur j cell biol 19 (1979) 109. 18. Osborn, M & Weber, K, Proc natl acad sci US 73 (1976) 867. 19. Porter, K R & Bonneville, M, Fine structure of cells and tissues. Lea & Febiger, Philadelphia, Pa (1973). 20. Reese, T S, J cell biol 25 (1965) 209. 21. Rieder, C L, Jensen, C G & Jensen, L, J ultrastruct res 68 (1979) 173. 22. Sandbom, E B, Cells and tissues by light and electron microscopy, vol. 1. Academic Press, New York. London (1970). 23. Satir,‘P, J cell diol 39 (1%8) 77. 24. Scherft, J P & Daems, W T, J ultrastruct res 19 (1967) 546. 25. Sorokin, S, J cell biol 15 (1962) 363. 26. Starger, J, Brown, W E, Goldman, A &Goldman, R D, J cell biol 78 (1978) 93. 27. Stephens, R, J cell bio164 (1975) 408. 28. Szollosi, D, J cell biol21 (1964) 465. 29. Telzer, B R & Rosenbaum, J L, J cell biol81(1979) 484. 30. Tilney, L G & Goddard, J, J cell biol46 (1970) 564. 31. Tucker, R W, Pardee, A B & Fujiwara, K, Cell 17 (1979) 527. 32. Warner, F D, Cell motility (ed R Goldman, T Pollard & J Rosenbaum) Cold Spring Harbor conferences on cell proliferation, -vol.-3. Cold Spring Harbor, New York (1976). 33. Wheatley, D N, J anat 105 (1969) 351. 34. - Ibid 113(1972) 83. 35. Zimmermann, K W, Arch mikr Anat 52 (1898) 552.
REFERENCES 1. Albrecht-Buehler, G, Cell 12 (1977) 333. 2. Albrecht-Buehler, G & Bushnell, A, Exp cell res 120(1979) 111.
Printed
in Sweden
Received July 13, 1979 Revised version received October 22, 1979 Accepted October 25, 1979
E,rl, Cd Rrs 126 ( 1980)
Experimental
Cell Research 126 (1980) 427-437
THE ULTRASTRUCTURE
OF PRIMARY
IN QUIESCENT GUENTER
3T3 CELLS
ALBRECHT-BUEHLER
Cold Spring Harbor
Laboratory,
CILIA
and ANNE BUSHNELL
Cold Spring Harbour,
NY 11724, USA
SUMMARY Electron microscopy was used to investigate primary cilia in quiescent 3T3 cells. As in the case of primary cilia of other cell types, their basal centriole was found to be a focal point of numerous cytoplasmic microtubules which terminate at the basal feet. There are also intermediate filaments which appear to converge at the basal centriole. Cross-striated fibers of microtubular diameter, reminiscent of striated rootlets of ordinary cilia, appear associated with the proximal end of the basal centriole. Usually less than nine cross-banded basal feet surround the basal centriole,in a well-defined plane perpendicular to the centriolar axis. The ciliary shaft was found to be enttrely enclosed in the cytoplasm of fully flattened cells. In rounded cells, it could be found extending to the outside of the cell. Periodic striations along the entire shaft were observed after preparing the cells in a special way. The tip of the shaft showed an electron-dense specialization. Several unusual forms of primary cilia were observed which were reminiscent of olfactory flagella or retinal rods. Using tubulin antibody for indirect immunofluorescence, a fluorescent rod is visible in the cells [18] which we demonstrate is identical with the primary cilium.
Primary (rudimentary, solitary) cilia and their biological function are still an enigmatic and often unknown aspect of animal cell physiology. They are defined as single cilia which grow out of one of the centrioles during interphase in otherwise unciliated animal cells. Before mitosis, the cilium shaft is disassembled [21] and only unciliated centrioles are found at the spindle poles [4, 291. Many of the primary cilia remain, for the most part, inside the cell body. In contrast to normal, motile cilia with the well-known 9+2 pattern, primary cilia show a 9+0 pattern [5]. Although discovered in 1898 [35], primary cilia were first carefully investigated in 1961 and 1962 by Barnes [5] and Sorokin [25], who described them in mouse hypophysis as well as in fibroblasts and smooth muscle cells of neonatal chicken. By the year 1967 Scherft
& Daems [24] listed 36 avian and mammalian tissues which contain primary cilia. In 1971 Wheatley [33, 341 described .such cilia in cultured cell lines such as BHK21 and 3T6. It seems, therefore, that primary cilia are quite common centriolar specializations in vivo and in vitro. According to the work of Sorokin [25] and Wheatley [33, 341 primary cilia form after a vacuole (ciliary vacuole) has attached to the distal end of one of the c,entrioles. Subsequently, the ciliary shaft elongates into the, vacuole. The shaft has a membrane around it which appears to be derived from the basal part of the ciliary vacuole. In many cases, the distal part of the elongated vacuole connects to the outside of the cell, thus leaving the now partially. external cilium located in a more or less deep basal cup. The connection Exp Cell Res 126 (19801
428
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of the ciliary vacuole to the outside does not always occur, especially in cultured cells. The biological function of primary cilia is unknown. Since retinal rods [8], olfactory flagellae [20] and kinocilia may be considered specializations of primary cilia, even though only retinal rods show a 9+0 pattern, one may suspect them of having a sensory function l-51, perhaps that of a chemoreceptor [16]. It was also noted that ciliation of centrioles correlated with a reduction of mitotic activity. Furthermore, it was found that blocking mitosis with colcemid induced primary cilia in CHO cells [7]. These findings led to the suggestion that primary cilia may be involved in control of the cell cycle [4, 9, 311, although this interpretation has been challenged [ 121. Furthermore, it was recently shown that PtKl cells resorb their primary cilium as late as during early spindle formation [21], which makes it less likely that primary cilia control cell division and more likely that they are subject to this control. Aside from these possibilities, using indirect immunofluorescence we recently found [l] that the primary cilia in migrating 3T3 cells was oriented parallel to the substrate and predominantly pointing in the direction of migration. In addition, we found in migrating 3T3 cells one centriole oriented preferentially parallel, the other perpendicular to the substrate [2]. The parallel centriole could be ciliated and, thus, easily observable by immunofluorescence. It seems that these observations add an aspect of spatial directionality to the suspected sensory function of primary cilia. In view of the difficulties of interpreting the biological function of primary cilia, it may seem appropriate to collect more information about them. Therefore, we studied the ultrastructure of primary cilia in quiescent 3T3 cells which express primary Exp Cell Res 126 (1980)
cilia at a frequency of 100% [l]. Several of our findings confirm previous descriptions of primary cilia in other cells. We include such features of the primary cilia of quiescent 3T3 cells in this report, nevertheless, because they may help to establish the common features of primary cilia.
METHODS Cell cultures. Swiss 3T3 cells, a kind gift of Dr Howard Green, Massachusetts Institute of Technology, were grown in Dulbecco’s modified Eagle’s medium (Grand Island Biological Co., Grand Island, N.Y .) supplemented with 10% calf serum (Microbial. Assoc., Bethesda, Md) in a 10% CO, atmosphere with saturated humidity. After reaching confluency at day 3, the cells were left in culture (without medium change) for another 7-9 days, in order to insure quiescence. Subsequently, they were processed for electron microscopy Immunojluorescence. Following a 30 min fixation of the cells in 3.5% formaldehyde in phosphate buffered saline (PBS), the cells were acetone extracted and incubated for 30 min at 37°C with rabbit antibovine brain tubulin antibody, a kind gift of Dr Frank Solomon, Massachusetts Institute of Technology.
Electron microscopy Preparation method I. Cells in dishes were fixed for 30 min with 2 % glutaraldehyde in 0.1 M phosphate buffer (PB) at DH 7.2-7.4. After nest-fixation for 30 min in 1% 0~6, in PB the cells were dehydrated in a graded ethanol-water series. Subsequently, the cells were left overnight under a layer of Epon 812 (Ted Pella Corp., Tuston, Calif.), reembedded in Epon 812, which was polymerized overnight at 70°C. The polymerized resin was popped out the dish, cut, trimmed, and sectioned with a diamond knife (DuPont Instruments, Wilmington, Del.) on an LKB ultrotome III. Specimens were examined with a Philips 201 electron microscope. Preparation method II. Same as I, except that cells were fixed in 2.5% glutaraldehyde in 0.1 M PB containing 0.1 M sucrose. Furthermore, before the cells were overlayed with Epon, they were left overnight in a 1 : 1 ethanol-Epon mixture.
RESULTS Centrioles and primary cilia in 3T3 cells Fig. 1 compares the ultrastructure of centrioles with that of primary cilia in 3T3 cells. Fig. 1b shows a typical longitudinal section through a centriole demonstrating the taper-
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Fig.
2. Comparison of normal centrioles (b-j’) with primary cilia of 3T3 cells (a, g-k) in longitudinal and cross-section. Bipointed arrows indicate approhimate location of the cross-sections. sl, Small central lumen;
ol, open lumen; bf basal feet; WC, ciliary vacuole. Arrowhead in h points to one of the alar sheets; arrowheads in e point to the electron-dense plaques which line 3 consecutive triplet blades. Bar, 0.5 pm.
ing of the wall microtubules toward the distal end. Two-thirds of the lumen of the centriole is filled with fibrous material beginning at the distal end. In axial sections (fig. lc-J) the fibrous material leaves a small central lumen (fig. 1d, e, “4”) which extends down the centriolar axis as far as the fibrous material. Near the proximal end the entire centriolar lumen appears empty. In 19 serial sections through centrioles in 3T3 cells we could not observe a cartwheel at the proximal end. Quite frequently electron opaque plaques appeared associated with the outer side of two or three consecutive blades near the proximal end (fig. 1e, arrowheads). In longitudinal section, the basal centriole of a primary cilium appears quite similar to a centriole (fig. la). However, basal feet which are not observed in unciliated centrioles (fig. 1a, “bf ‘) and which
are the focal point of numerous microtubules appear connected to the wall. They will be described in more detail below. In fig. 1a, we used preparation method II, for better visibility of the ciliary vacuole (fig. la, g, “vat”). Near the bottom the membrane of the ciliary vacuole appears quite dense (see also the other figures of this paper). A periodic striation extending up the shaft can be observed (see also below) indicating the presence of a ciliary necklace as in normal cilia. In axial section (fig. 1g-k) the basal centriole appears quite similar to the normal centriole. However, in addition to the basal feet (fig. 1i, “bf’), transitional fibers or alar sheets (fig. lh, arrowhead) [3] can be observed, as in ordinary cilia. Above the distal end, the section through the ciliary shaft (fig. 1g) shows the usual microtubular doublet array in the 9+0 pattern. The ciliary membrane and the Exp Cell Rrs 126 (1980)
Fig. 2. Identification of the primary cilium in immunofluorescence using tubulin antibody. (The long dark line in c is a fold in the section.) The panels show the same 3T3 cell in phase-contrast (a); indirect immunofluorescence (b; arrow points to the fluorescent rod); transmission electron microscopy (ventral, c;
higher up, d; arrow points to primary cilium in identical position and orientation as the fluorescent rod). e, Primary cilium at higher magnification. All microtubules are destroyed due to the preparation for immunofluorescence. Bar, (a, d) 20 pm; (e) 1 pm.
membrane of the ciliary vacuole in crosssection are usually separated.
[18] to reveal a rod-like fluorescent structure which appeared to be a nucleation center of cytoplasmic microtubules. Osborn & Weber, as well as subsequent investigators [l, 311, interpreted this rod as the primary cilium. The formal proof for this identification has not yet been given. Fig. 2
Primary cilia in indirect immunofluorescence Indirect immunofluorescence using tubulin antibody was shown by Osborn & Weber Exp
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Fig. 4. Details of the basal feet structure. (u-d) Grazing sections through the tip of basal feet demonstrating their planar arrangement in a quite reproducible position. Arrows point to portions of the sectioned basal feet; (e-i) cross-banding of single basal feet (arrowheads). (g) One of the observed basal centrioles with two basal feet (arrows) above each other. Bar, 0.5 pm.
Fig. 3. Primary cilium of a post-telophase 3T3 cell extending to the outside of the cell. Bar, 1 pm.
attempts to close this gap by comparing the fluorescence micrograph and the electron micrograph of the same 3T3 cell. Beside the phase micrograph of a 3T3 cell (fig. 2a) fig. 2b shows the fluorescent micrograph
of the same cell with the fluorescent rod. Before this micrograph was taken, the cell had been formaldehyde-fixed, acetoneextracted, and fluorescence-stained. Subsequently, the cell was post-fixed with glutaraldehyde and osmium tetroxide and processed for electron microscopy. Fig. 2c shows a ventral section parallel to the substrate. The nucleoli pattern, general shape of cell and nucleus demonstrate the identity between this cell and the one photographed in phase contrast. In a higher section (fig. 2 d) the primary cilium appears in the identical location and orientation as the fluorescent rod. At higher magnification (fig. 2e) it is apparent that the formaldehyde-acetone treatment destroyed all microtubules. Nevertheless, basal centriole, ciliary vacuole, shaft and necklace of the structure can clearly be distinguished. Electron microscopy
of primary cilia
The following description is based on approx. 600 electron micrographs of primary cilia in quiescent 3T3 cells which were Exp CellRes 126 (1980)
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Fig. 5. Grazing section through two basal feet of a primary cilium. The shaft (recognizable by the crossstriations) extends in the direction of the arrow. The
section illustrates the elaborate radial organization of cytoplasmic microtubules (mt), but also of intermediate filaments (iJ) around primary cilia. Bar, 1 ,um.
sectioned parallel to the substrate. With only a few exceptions, it extended parallel to the substrate. In all flattened cells we found the cilium to be entirely inside the cytoplasm. Serial sections as well as numerous single sections showed the primary cilium surrounded from all sides by cytoplasm (see, e.g. fig. 9). In still rounded, reflattening, post-telophase cells, however, we found several cases of cilia which extended to the outside of the cell (fig. 3). Most tissue cells in vivo have a more rounded shape than flattened cultured cells. Since their primary cilia in most cases were found to emerge to the outside [4, 12, 24,
251, it seems possible that the complete enclosure of primary cilia in 3T3 cells is caused by their flattening on the artificial substrate. The basal centriole of primary cilia was found to have a diameter of 0.26 pm and lengths between 0.41 and 0.51 pm. In 27 sections which showed the entire ciliary shaft we found a shaft length of 4 pm (S.D. 1.5 pm). However, there were cilia as long as 7 pm. The primary cilium was usually found near a nuclear indentation. The distance between the basal centriole and the nuclear envelope was measured in 130 micrographs which showed the cilium to-
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Fig. 6. Striated “rootlets” (arrows) of about 250 A diameter connected to basal centrioles. Bar, 0.5 pm.
gether with the nucleus and yielded a surprisingly constant distance of 1.2 pm (S.D. 0.5 pm). A constant distance between basal centriole and nuclear envelope seems to support the suggestion of a material connection between them [6, 171. We were unable to find an obvious connection in the micrographs. However, it may be possible to observe it only after extraction of the cytoplasmic matrix as in the quoted experiments. The two centrioles of a pair are usually oriented at right angles to each other. In sections of quiescent 3T3 cells which showed the second unciliated centriole, we rarely found this to be the case. Most of the time, the associated centriole was at some oblique angle relative to the axis of the basal centriole of a primary cilium. Basal feet
The most striking ultrastructural feature of primary cilia is the convergence of cytoplasmic microtubules [30] toward the basal feet [15]. The focussing of cytoplasmic microtubules towards primary cilia has also
Fig. 7. primary primary also fig.
Dense tip specializations of three different cilia (cf also fig. 3). Arrows in c show two cilia enclosed in the same ciliary vacuole (cf 9e). Bar, 0.5 pm.
been demonstrated by indirect immunofluorescence [ 181.The basal feet are located within a plane collar around the centriolar wall. This is apparent in axial sections through this collar or even better, in grazing longitudinal sections which cut through the tips of the basal feet such as shown in fig. 4a-d. In three exceptional cases, however, we found two basal feet, one above the other, in the axial direction (fig. 4g, arrowheads). Fig. 4 also illustrates that the collar of basal feet is located at a rather reproducible distance from the proximal end. Measurements of 57 sections showed this distance to be 0.3 pm (S.D. 0.1 pm). Confirming earlier observations [ 10, 281, we found that basal feet have a conical shape with a triple cross-banding (fig. 4e-i, arrowheads). It may also be noteworthy that in three perfectly axial cross-sections through the collar of basal feet we found less than 9 basal feet surrounding the centriolar wall (see also fig. 1i). Exp Cell Res 126 (1980)
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around the basal centriole yields an estimate of 130 microtubules which converge to one basal centriole. Assuming that the second centriole of a pair is surrounded by equally many microtubules, one would estimate that 260 microtubules converge to the centriole pair. “Rootlets”
Striated fibers were regularly observed near primary cilia. Most of them appeared to arise from the proximal end of the basal centriole. Some cases showed quite elaborate arrays of these fibers (fig. 6a) or even a large loop (fig. 6b). The visibility of the striated fibers was hampered by the surrounding cytoplasmic matrix which made it impossible to follow them through serial sections. Therefore, we do not know where they terminate in the cell. They showed a diameter of 250 A and a periodicity of 730 A (S.D. 20 A) and appeared significantly difFig. 8. Periodic shaft striations observed in primary ferent from the striated rootlets in ordinary cilia using fixation method II. Bar, 0.5 pm. cilia [3, 271, which have a periodicity of 650 A and a diameter of 700-800 A. Since The basal feet are the focal point of an we never observed any other kind of strielaborate array of cytoplasmic microtu- ated “rootlets”, it is not clear whether these bules (fig. 5). As illustrated in fig. 6, there structures are rootlets in the sense of ordiare also numerous intermediate filaments nary cilia. The diameter of 250 8, of the which appear to run parallel to microtu- striated rootlets suggests the possibility that bules towards the basal centriole. In addi- they are special microtubules which are tion to these fibrous structures, numerous periodically labelled with associated comvesicles were observed surrounding the ponents. cilia. Occasionally, we found the previously described [13] virus-like particles near the The ciliary shaft basal centrioles. At the base of the ciliary shaft the expected It is apparent from fig. 5 that the basal 9+0 doublet pattern is quite regular. Higher centriole is a major organization center for up in the ciliary shaft the 9 microtubular cytoskeletal fibers. This micrograph shows doublets appear to become disorganized. about 35 microtubules converging to the Occasionally we saw material connections basal centriole. With a section thickness of between the ciliary shaft and the surroundabout 1000 A one can estimate the average ing ciliary vacuole and even unidentified spatial sector occupied by one microtubule. inclusions. The latter were only seen in Extrapolating this value to an entire sphere highly vacuolarized cells suggesting the Exp CellRes 126(1980)
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Fig. 9. Unusual forms of primary cilia. (a, b) Bulging shafts with dense inclusions; (c) enclosed large vacuole; (d) bulging dense tip; (e) two parallel cilia
in the same ciliary vacuole; Bar indicates 0.5 pm. (f) unusually long shaft. Bar, (a-e) 0.5 pm; (f) 1 pm.
possibility that these were the result of a degeneration process. Whenever the sections contained the tip of the ciliary shaft, we observed electrondense material concentrated in the tip, demonstrating that the organization of the ciliary shaft is not uniform along its length. Fig. 7 shows three different examples which illustrate how reproducible this tip specialization is. A similar tip was shown in fig. 3. The tip density could become quite expanded with included vacuoles and even dense substructures. Fig. 9d shows such a bulging tip which is quite similar to a micrograph of Sorokin’s [25] obtained from a fibroblast in organ culture. Tip densities are not observed in ordinary cilia of vertebrates
primary cilia after cells recovered from colcemid treatment. We found 27 cases of similar striations extending along the entire ciliary shaft after the cells had been treated by preparation method II (fig. 8). Using the normal preparation I, we found such extended striations in only two cases, although a short stretch of striations is generally observed at the base of the ciliary shaft. In the literature, these short stretches have been termed the ciliary necklace. Preparation method II, which obviously destroys all membranes, including that of the ciliary vacuole and shaft, may facilitate the observation of the ciliary necklace extending all the way up the ciliary shaft. Measuring the periodicity of the striations in 17 cilia over at least 8 periods each we found a periodicity of 430 8, (SD. 30 A). In ordinary cilia the dynein repeat is 143 A and the spokes repeat with a periodicity of 860 8, every three unequally spaced spokes
[231. Shaft striations In several cases, Archer & Wheatley [4] observed a conspicuous cross-striation of
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[32]. None of these periodicities coincides with the periodicity of the shaft striations. Therefore, it seems unlikely that these striations are related to the spokes or dynein organization of ordinary cilia. It should be noted, though, that three dynein repeats would generate 430 A. It is also noteworthy that two periods of 430 A would generate the 860 8, of the spokes repeat. Interestingly, the outer rod segments of retinal rods are spaced by 400 A [ 191,which is the same distance as the observed shaft striations. Unusual structures of primary
cilia
Several primary cilia showed quite unusual ultrastructures. Some of them are shown in fig. 9. Unusually long ciliary shafts (fig. 9f, and the enlarged dense tip shown in fig, 9d have been mentioned above. Confirming other observations on different cells [5, 341 we found several cases with two parallel cilia enclosed in the same ciliary vacuole (figs 7 c , 9 e). Furthermore, we found several cases with dramatically enlarged and bulging ciliary shafts (fig. 9a, b) including cases with large enclosed vacuoles (fig. SC). More frequently, small vacuoles were observed inside the shaft as well as dense bodies such as seen in the lefthand side of fig. 9a. Such cases were observed using normal preparation method I as well as method II. Nevertheless, the possibility cannot be excluded that preparation artifacts or degeneration processes in the quiescent cells produced these unusual forms. We mention their existence for the sake of completeness. DISCUSSION The present study describes primary cilia in quiescent 3T3 cells as elaborately structured organelles quite similar to primary E.l-l, Cell Res 126 (1980)
cilia in other cell types. Their basal centrioles are a focal point of cytoplasmic microtubules, but apparently also of intermediate fdaments, and of striated fibers of microtubular diameter, which may be related to the striated rootlets in ordinary cilia. There have been earlier suggestions of an association between intermediate filaments and centrioles. The caps of intermediate filaments in spreading BHK21 cells contain centrioles [26]. A rather striking association between centrioles and intermediate filaments has been observed in duodenal epithelium [22]. In this case a tight ring of such filaments encircles the centriole in a plane perpendicular to its axis. It appears that basal centrioles of primary cilia bring the converging cytoplasmic microtubules closer to the centriolar wall than normal centrioles. Although normal centrioles as seen in electron microscopy of whole cells are more or less radially surrounded by cytoplasmic microtubules, the microtubules rarely seem to come closer than 0.5-l pm to the centriolar wall. In contrast, isolated centrioles can nucleate microtubules at the wall [13, 141 which is surrounded by the so-called pericentriolar material. Telzer & Rosenbaum, using centrioles of HeLa cells, have recently shown that the microtubule nucleating capacity of isolated centrioles is most pronounced in centrioles from mitotic cells [29]. Considering the rather spectacular microtubular organization around basal centrioles of primary cilia in quiescent 3T3 cells, one may suggest that GO presents another phase of the cell cycle where centriolar nucleation of cytoplasmic microtubules is enhanced. The observation that less than nine basal feet can be found around the wall of the basal centriole, but also the observation of 2-3 electron-opaque plaques on consecu-
Primary cilia in 3T3 cells tive blades in centrioles, suggest that the nine blades of a centriole are not equivalent. It is tempting to speculate that such asymmetries in the otherwise so perfectly symmetrical centrioles are important for the postulated role of centrioles in controlling cell migration or other directional functions of cells. The cross-striations along the ciliary shaft which appear to be an extension of the ciliary necklace may indicate that some, if not all, primary cilia amplify a biological function of the ciliary necklace. It appears that the ciliary necklace binds calcium [ 111. P. Satir has suggested (personal communication) that the shaft of the primary cilium may serve as a calcium source or sink. The ciliary shaft is isolated from the rest of the cytoplasma either by the ciliary vacuole or because it extends to the outside. In either case, it appears to be accessible only through the basal centriole. Consequently, one may contemplate a directional release of calcium from the shaft into the cell with all its ramifications as to calcium-sensitive cellular and molecular processes which it may render directional. It remains to be seen whether the shaft of the primary cilium binds calcium in a similar way as ordinary cilia do. We are grateful for Dr James D. Watson’s support. The photographic work of Mr Ted Lukralle and the skillful typing of MS Madeline Szadkowski are gratefully acknowledged. The work was supported by the Cold Spring Harbor Laboratory Cancer Center grant from the NCI.
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3. Anderson, R G W, J cell biol54 (1972) 246. 4. Archer, F L & Wheatley, D N, J anat 109 (1971) ^-LII. 5. Barnes, B G, J ultrastruct res 5 (1961) 453. 6. Bornens, M, Nature 270 (1977) 80. 7. Brinkley, B R & Stubblefield, E, J ultrastruct res 19 (1967) 1. 8. DeRobertis, E, J biophys biochem cytol 2 (1956) 319. 9. Dingemans, K P, J cell bio143 (1969) 361. 10. Doolin, P F & Birge, W J, J cell biol29 (1966) 333. II. Fisher, G, Kaneshiro, E S & Peters, P D, J cell biol69 (1976) 429. 12. Fonte, V G, Searls, R L & Hilfer, S R, J cell biol 49 (1971) 226. 13. Gould, R R & Borisy, G G, J cell bio173 (1977)601. 14. Heidemann. S R. Sanders. G & Kirschner. M W. Cell 10 (1977) 337. 15. Kalnins, V I & Porter, K R, Z Zellforsch 100 (1963) 1. 16. Muneer. B L. Am i anat 103 (1958) 275. 17. Nadizhdina,‘E S, “Fais, D & ‘Chentsov, Y S, Eur j cell biol 19 (1979) 109. 18. Osborn, M & Weber, K, Proc natl acad sci US 73 (1976) 867. 19. Porter, K R & Bonneville, M, Fine structure of cells and tissues. Lea & Febiger, Philadelphia, Pa (1973). 20. Reese, T S, J cell biol 25 (1965) 209. 21. Rieder, C L, Jensen, C G & Jensen, L, J ultrastruct res 68 (1979) 173. 22. Sandbom, E B, Cells and tissues by light and electron microscopy, vol. 1. Academic Press, New York. London (1970). 23. Satir,‘P, J cell diol 39 (1%8) 77. 24. Scherft, J P & Daems, W T, J ultrastruct res 19 (1967) 546. 25. Sorokin, S, J cell biol 15 (1962) 363. 26. Starger, J, Brown, W E, Goldman, A &Goldman, R D, J cell biol 78 (1978) 93. 27. Stephens, R, J cell bio164 (1975) 408. 28. Szollosi, D, J cell biol21 (1964) 465. 29. Telzer, B R & Rosenbaum, J L, J cell biol81(1979) 484. 30. Tilney, L G & Goddard, J, J cell biol46 (1970) 564. 31. Tucker, R W, Pardee, A B & Fujiwara, K, Cell 17 (1979) 527. 32. Warner, F D, Cell motility (ed R Goldman, T Pollard & J Rosenbaum) Cold Spring Harbor conferences on cell proliferation, -vol.-3. Cold Spring Harbor, New York (1976). 33. Wheatley, D N, J anat 105 (1969) 351. 34. - Ibid 113(1972) 83. 35. Zimmermann, K W, Arch mikr Anat 52 (1898) 552.
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Printed
in Sweden
Received July 13, 1979 Revised version received October 22, 1979 Accepted October 25, 1979
E,rl, Cd Rrs 126 ( 1980)