Vertebrate Primary Cilia: a Sensory Part of Centrosomal Complex in Tissue Cells, but a “Sleeping Beauty” in Cultured Cells?

Cell Biology International Cell Biology International 28 (2004) 139–150 www.elsevier.com/locate/cellbi

Vertebrate primary cilia: a sensory part of centrosomal complex in tissue cells, but a “sleeping beauty” in cultured cells?

Irina B. Alieva*, Ivan A. Vorobjev

Laboratory of Cell Motility, A.N. Belozersky Institute for Physico-Chemical Biology, Moscow State University, Moscow, 119992, Russia

Received 22 July 2003; accepted 4 November 2003

Abstract

Primary cilium development along with other components of the centrosome in mammalian cells was analysed ultrastructurally and by immunoﬂuorescent staining with anti-acetylated tubulin antibodies. We categorized two types of primary cilia, nascent cilia that are about 1 µm long located inside the cytoplasm, and true primary cilia that are several µm long and protrude from the plasma membrane. The primary cilium is invariably associated with the older centriole of each diplosome, having appendages at the distal end and pericentriolar satellites with cytoplasmic microtubules emanating from them. Only one cilium per cell is formed normally through

G0, S and G2 phases. However, in some mouse embryo fibroblasts with two mature centrioles, bicilates were seen. Primary cilia were not observed in cultured cells where the mature centriole had no satellites and appendages (Chinese hamster kidney cells, line 237, some clones of -fibroblasts). In contrast to primary cilia, striated rootlets were found around active and non-active centrioles with the same frequency. In proliferating cultured cells, a primary cilium can be formed several hours after mitosis, in fibroblasts 2–4 h after cell division and in PK cells only during the S-phase. In interphase cells, formation of the primary cilium can be stimulated by the action of metabolic inhibitors and by reversed depolymerization of cytoplasmic microtubules with cold or colcemid treatments. In mouse renal epithelial cells in situ, the centrosome was located near the cell surface and mature centrioles in 80% of the cells had primary cilium protruding into the duct lumen. After cells were explanted and subcultured, the centrosome comes closer to the nucleus and the primary cilium was depolymerized or reduced. Later primary cilia appeared in cells that form islets on the coverslip. However, the centrosome in cultured ciliated cells was always located near the cell nucleus and primary cilium never formed a characteristic distal bulb. A sequence of the developmental stages of the primary cilium is proposed and discussed. We also conclude that functioning primary cilium does not necessarily operate in culture cells, which might explain some of the contradictory data on cell ciliation in vitro reported in the literature. 2003 Elsevier Ltd. All rights reserved.

Keywords: Centrosome; Centrioles; Primary cilium; Striated rootlets; Pericentriolar satellites; Microtubules

1. Introduction cilia. The primary cilium is non-motile organelle that occurs singly on most cells in the vertebrate body that Formation of primary cilium is one of the basic have been carefully examined, with the few exceptions functions of the centrosome (Barnes, 1961; Sorokin, being cells of myeloid and lymphoid origin (Wheatley, 1962). Primary cilium is a special structure forming on 1995). Primary cilia are frequently found in embryonic the centriole. It has 9+0 axoneme that means nine tissues and during early postnatal development (Fonte doublets of microtubules with no microtubule in the et al., 1971; Jurand, 1974; Sorokin, 1962; Tucker et al., center. Primary cilium consists of two parts—a basal 1992). Also many diﬀerentiated cells have primary cilia body and a membrane-bound ciliary axoneme that lacks (Albrecht-Buehler, 1992; Bystrevskaya et al., 1992; the central doublet and dynein arms typical for motile Dahl, 1963; Dalen, 1981; Latta et al., 1961; Poole et al., 1985; Wheatley et al., 1994). There are few cell types * Corresponding author. Tel: 7-(095)-939-5528; where a primary cilium is not formed, e.g. nucleated Fax: 7-(095)-939-3181 blood cells, adipocytes, hepatocytes (Wheatley, 1969, E-mail address: [email protected] (I.B. Alieva). 1982).

Primary cilia were found in different cultured fibro- medium well provided for constancy of environment and blasts (Albrecht-Buehler, 1977; Tucker et al., 1979a,b; surroundings. However, it might be possible that in Wheatley, 1971; Wheatley et al., 1994, 1995) where up to cultured cells, primary cilium has no special function or 87–88% of cells might be ciliated (Wheatley, 1982). In its function is not permanent. The percentage of ciliated the epithelial cell lines percent of ciliated cells is cells in vitro is different and can differ from one passage lower—about 30% (Alieva and Vorobjev, 1989; to another (Alieva et al., 1999). The highest percentage Bystrevskaya et al., 1992; Rieder et al., 1979; Roth et al., of ciliation has always been observed among non- 1988; Wheatley, 1971). proliferating cells (Tucker and Pardee, 1979; Wheatley, Although functions ranging from vestigial remnants 1995). In the current study, we have analyzed the details to sophisticated sensory devices such as the rods and of primary cilia structure in several cell types, changes in cones of the retina have been attributed to primary cilia, the primary cilium during cell culturing and spreading their function in a variety of cell types has not been on the substrate in vitro, primary cilia formation during ascertained (Poole et al., 1997; Wheatley, 1995). A the cell cycle, and the reaction of primary cilia to sensory function of the primary cilium was proposed different metabolic inhibitors. long ago (Poole et al., 1985; Roth et al., 1988; Schwartz et al., 1997); however, only recently have experimental 2. Materials and methods findings tended to confirm this hypothesis. It was sug- gested that the primary cilium operates as part of a 2.1. Cell cultures sensory pathway, translating extracellular information to the centrosome and the Golgi complex to facilitate Epithelial PK (pig kidney) cells, Chinese hamster the efficient secretion of newly synthesized material to kidney cells, and -fibroblasts were cultured at 37 (C the extracellular matrix (Poole et al., 1997). Thus the and 5% CO2 on medium 199 supplemented with 10% primary cilium serves an antenna, displaying specific bovine serum and gentamycin. HeLa cells were grown at ( receptors to the body in different tissues, e.g. kidney 37 C and 5% CO2 in medium 199 supplemented with primary cilia display polycystin-2, which forms part of a 10% fetal calf serum and gentamycin. 3T3 cells, rat 2+ Ca channel initiating a signal that controls cell differ- kangaroo PTK1 cells and primary rat embryo fibroblasts ( entiation and proliferation (Pazour et al., 2002a,b; (REF) were grown at 37 C and 5% CO2 in DMEM/ Somlo and Ehrlich, 2001). Kidney primary cilia also F-12 HAM (Sigma, USA) supplemented with 10% fetal serve as mechanosensors that, when bent, initiate a calf serum and gentamycin. Ca2+ influx that spreads throughout the cell and to Primary cultures of mouse fibroblasts (MEF) and the neighbouring cells (Pazour and Witman, 2003; renal epithelial cells were obtained from 15–19 day Praetorius and Spring, 2001). Primary cilia in the other mouse foetuses according to the standard protocol. Cells ( cell types specifically display different tissue-specific were cultured at 37 C and 5% CO2 on medium 199 receptors, including those for somatostatin (Handel supplemented with 10% fetal calf serum and gentamycin et al., 1999) and serotonin (Brailov et al., 2000)in and analysed in the first passage. neuronal cilia. 2.2. Drug and cold treatments Since primary cilium serves as an antenna enhancing specific external signals, it is natural to assume that All drugs used were added to the culture medium centrosome at the base of primary cilium could be from stock solutions at the following final concen- involved in the signal transduction pathway. The major trations: FCCP—20 µM; 2,4-dinitrophenol (DNP)— function of the centrosome is formation of radial micro- 800 µM; sodium azide—20 mM, ouabain—1 mM, cal- tubule array, and activity of the centrosome in the cium ionophore A23187—20 µM. Colcemid was added organization of microtubules is variable. We suggest at a final concentration of 10 µg/ml. Cold treatment was that increased formation of microtubules on the centro- performed as described elsewhere (Vorobjev and some could be induced by initiation of the signal trans- Chentsov, 1983). Recovery from colcemid treatment was duction from primary cilium and examined correlation performed by triple change of the culture medium to the between centrosome activity and frequency of primary fresh one. Recovery from the cold treatment was by cilium formation. transfer of the Petri dishes with cells from the ice-bath to In general, sensory function of the primary cilium in the 37 (C incubator. tissue (in situ) seems to be arguable and clarified, but several questions regarding ciliogenesis and cilia remain 2.3. Determination of the phase cell cycle to be answered. It is very difficult to inquire into these questions with tissues in situ, but much more easy to Determination of the stages of cell cycle was per- make experiments using cultured cells. But the primary formed for individual cells using double H3- and C14- cilia functions are absolutely unclear in cultured cells, thymidine labelling as described elsewhere (Vorobjev when tissue lost its 3D organization and cultural and Chentsov, 1982). Cells were attributed to G0 period I.B. Alieva, I.A. Vorobjev / Cell Biology International 28 (2004) 139–150 141 when they did not incorporated labelled thymidine for at rpm three times 3 min each; during centrifugation least 24 h. the cells were washed with the medium without nocodazole. The sedimented metaphase cells were 2.4. Antibodies plated in a drop of the medium on to coverslips. 30 min after (upon complete attachment of the cells to Monoclonal antibodies C3B9 against acetylated the cover-slips) 3 ml of the medium was added to the tubulin were produced at the Laboratory of Keith Gull Petri dishes. (Manchester, UK). Monoclonal antibodies against 2. Mitotic 3T3 cells were collected by shaking off -tubulin were obtained at the Laboratory of I.A. normal metaphase cells and plating them on to Vorobjev and described earlier (Komarova et al., 1996). coverslips as above but without nocodazole treat- FITC-conjugated secondary antibodies were obtained ment. Thus, no mitostatic was used in this case and, from Sigma (USA). by excluding its washout from the protocol, we 2.5. Immunofluorescent studies reduced the time from the beginning of isolation to the time when cells were attached to coverslips. For immunofluorescent analysis, the cells were fixed using two techniques: (i) in 4% formaldehyde solution for 30 min; (ii) in 1% glutaraldehyde solution (Merck, 3. Results Germany) in phosphate buffer for 30 min followed by a triple treatment with a NaBH4 solution (2 mg/ml, 10 min each). In the latter case, prior to fixation the cells were Electron microscopy and immunofluorescent study of lyzed in a mixture containing 1% Triton X-100 in the centrosome structure and appearance of primary ff microtubule-stabilizing conditions. The coverslips with cilia was analyzed in di erent cultured cells. The major the cells were taken out of the Petri dish, washed several part of this study was performed on kidney epithelial times with phosphate buffered saline (pH 7.2) at 37 (C cells (PK cells and primary mouse embryo kidney cells), and then lyzed for 15 min in a solution containing and in some studies we used other cell types. 50 mM imidazole (pH 6.8), 5 mM MgCl ,1 mM EGTA, Using serial EM sections we found nascent primary 2 ff 0.1 mM EDTA, 35% glycerol and 1% Triton X-10 cilia in di erent cultured cell lines including fibroblasts (Sigma). The preparations were included into a 2.5% and epithelial cells and also in primary cultures. The solution of 1,4-diazabicyclo-[2.2.2]-octane (DABCO) minimal cilium determined using serial ultrathin sections (Sigma) on glycerol, examined under an Opton-3 photo- was about 0.5 µm long and hardly could be detected microscope (Opton, Germany) and photographed (film under the light microscope. However, detailed examin- RF-3, Tasma, Russia). ation of the centrosome confirmed the absence of primary cilium in blood cells (monocytes and macrophages, 2.6. Electron microscopy lymphocytes and neutrophils). Also we failed to find primary cilium in those cells where two centrioles were Cells were fixed with a 2.5% solution of glutaralde- of the same structure and capacity in formation hyde (Merck, Germany) in 0.1 M phosphate buffer (pH of cytoplasmic microtubules. These were epithelioid 7.2–7.4) for 30 min or lyzed in a mixture containing Chinese hamster kidney cells and -fibroblasts. Triton X-100, in microtubule-stabilizing conditions, and In all cell types analyzed where the primary cilia were then fixed with 1% solution of glutaraldehyde (Merck) in found, their fine structure was conservative—forming on the same buffer for 30 min (Alieva and Vorobjev, 1995). the centriole cylinder. It has axoneme of 9+0 formula, Further preparation for electron microscopy was per- which means nine doublets of microtubules with no formed as described earlier (Alieva and Vorobjev, 1995). microtubules in the center. The length of individual Serial ultrathin sections were made through the centro- doublets was different, and in apical cross-sections some region and examined under HU-11B or H-700 primary cilia have fewer of them (between 7 and 3). electron microscopes as described elsewhere (Alieva Formation of the primary cilium was always et al., 1992). restricted to the mature centriole. In cells with two centrioles there is only one primary cilium; but in cells in 2.7. Synchronization of cells G2 period where only one of four centrioles was mature also only one cilium formed. However, in some primary Mitotic cells were isolated by two techniques: cultured murine fibroblasts with two pairs of centrioles, 1. Mitotic cells of a rat fibroblast culture were accumu- two cilia were found (Fig. 1). This is in accord with the

lated by incubation of a monolayer culture in a data on primary cilia cycle in PtK1 cells: cells released medium containing nocodazole (0.1 µg/ml) for 4–6 h. from a continuous taxol treatment contained two nor- Then the metaphase cells were shaken oﬀ, collected mal centriole pairs, and one or both mature centrioles into 2-ml Eppendorf tubes and centrifuged at 1000 possess primary cilium (Jensen et al., 1987). 142 I.B. Alieva, I.A. Vorobjev / Cell Biology International 28 (2004) 139–150

Fig. 1. Electron micrograph of serial sections (1, 3, 6 and 9) showing the ﬁne structure of the centrosome and two ciliated active centrioles in MEF cell. A–D—two active centrioles with primary cilia (on the sections 1, 3 and 6) and two nonactive centrioles (on the sections 1, 6 and 9). A—active centriole; N—nonactive centriole, PC—primary cilium. Bar=0.5 µm.

3.1. Primary cilium in renal epithelium In isolated and suspended renal cells the centrosomes To compare primary cilia in situ and in cultured cells were located near the cell nucleus and the percentage of we used renal epithelium from mouse embryo. In the ciliated cells decreased. renal duct cells centrosome is located near the cell apex Renal epithelial cells spread slowly on the coverslip and far away from the nucleus. Two centrioles had and after several hours formed islets among fibroblasts. structure typical for the centrosome in interphase cells: During cell spreading centrosome was always located in one mature centriole had nine appendages at its distal the vicinity of cell nucleus and 30 min after seeding with end and often (14 out of 18 cells examined on serial the same probability it was under the nucleus above it or sections) formed primary cilium that was several mi- aside. Nascent primary cilia were found in 30% of cells. crons long and directed into the duct lumen. Cilium had These cilia were embedded into the cytoplasm and never axoneme 9+0 and often formed a bulb at the distal end came to the cell surface. In the single epithelial cells (not shown). The daughter centriole was located in the that had no contacts with neighbours the same picture vicinity of the mature one but had no appendage and no retained up to 24 h after seeding—primary cilium was pericentriolar satellite. Both centrioles were embedded in found in 30% of cells, it was very short and did not the fine fibrillar matrix. appear on the cell surface. I.B. Alieva, I.A. Vorobjev / Cell Biology International 28 (2004) 139–150 143

In the cells that form islets centrosome and primary of cases. These cilia were 2–3 µm long or more and cilia changed. In the internal cells of the islet cilia first always came out to the cell surface (Figs. 2 and 3). Thus appeared on the cell surface 6 h after seeding. When cell in PK cells primary cilia appears after rather long period islets were completely stretched on the coverslip location after cell goes into the interphase—G1 period in these of centrosome and primary cilia in internal cells and cells lasts for >5 h. To determine formation of the marginal cells of the islets became different. In the primary cilia more precisely we used epithelial cells and marginal cells centrosome was always located aside of fibroblasts synchronized in mitosis and analyzed pri- the nucleus between nucleus and free cell edge. 70% mary cilia formation by immunofluorescent staining of the marginal cells had short primary cilium (with of cilia with antibodies against acetylated tubulin axoneme 0.5–1 µm long), but it was always embedded (Wheatley et al., 1994, 1996). into the cytoplasm. In the internal cells centrosome was also close to the cell nucleus but located near the plasma 3.3. Formation of the primary cilium after mitosis membrane of the upper cell surface. 80% of cells in the Since primary cilium might be rather short and hardly islets had primary cilium that was usually found on the visible under the light microscope, to validate the upper cell surface. This cilium was 2–5 µm long, but has method of immunostaining we first compared frequency no bulb on its distal end. Thus primary cilia restored in of primary cilia in epithelial and fibroblast cell cultures the renal cells inside the islets, but these cilia were of using EM serial sectioning and immunostaining (Table different fine structure from the original ones. 1). The two approaches appear to be in a very good The next question was about behavior of the primary accord with each other. cilium during the cell cycle. It was shown that primary We further analyzed formation of primary cilium cilium is lost when cell enters mitosis (Jensen et al., 1987; after mitosis in two types of cultured fibroblasts. In Rieder et al., 1979), and we addressed the question when primary culture of rat embryo fibroblasts mitotic cells it appears after completion of cell division. Addressing were accumulated using low dose of nocodazole this question we used two approaches: first, cells were (0.1 mg/ml for 4 h). One hour after mitotic cells were individually selected at different stages of the cell cycle collected and plated on to the coverslip telophase cells and second, mitotic cells were collected, subcultured and and pairs of cells connected with residual body were examined at different time point after subculturing. observed. No primary cilium was found at this time. First cilia appeared 2 h after cell plating and later on 3.2. Primary cilium in the cell cycle their percentage slightly increased (Table 2). Taking into account that nocodazole might perturb formation of In some cultured cells structure of mature centriole primary cilium in the next set of experiments we used is different from regular one described elsewhere 3T3 cells where mitotic cells could be collected by (Vorobjev and Chentsov, 1980, 1982). The difference is shaking without treatment with nocodazole or other in the absence of appendages at the distal end and mitotic poison. In mitotic cells plated on the coverslip no pericentriolar satellites. Consequently, cytoplasmic cilium was found 1 h after plating, low percent of microtubules radiate in the same way from both centri- ciliated cells appeared 2 h after plating, and after4hof oles in such centrosome. We observed these “incom- culturing percentage of ciliated cells was the same as in plete” centrioles in -fibroblasts and Chinese hamster control culture. Occasionally 4 h after seeding primary kidney cells, line 237. In both cultures we failed to find cilia were formed in both sister cells, but mainly it primary cilium (no cilium for 80 cells in each culture). appeared only in one cell from the pair. In all other cases primary cilium was one per centriole Formation of primary cilium after cell division was pair and it was associated with the mature centriole. In found to be a rather long process and percentage of some cells in primary fibroblast culture (MEF) there ciliated cells was different from one cell type to another. were four centrioles and they had two primary cilia The next question is whether primary cilium can form in (Fig. 1). response to different treatments. In the overall population of PK cells (in regular culture conditions) about 10% of centrosomes had 3.4. Primary cilia and centrosome activity primary cilium. Analyzing individual cells at different periods of the cell cycle we found that in PK cells It was shown that a variety of drugs, including primary cilium is absent in G1 period. It first appears calcium ionophore A23187, uncouplers of oxidative only in S-phase (in 10% cases—3 of 28 cells) and became phosphorylation DNP, FCCP, treatment with sodium + + more frequent in G2 (37%—6 of 16 cells). These cilia azide and inhibitor of K /Na ATPase ouabain can were always short (with axoneme about 1 µm long) and “activate” the centrosome, causing an increase in the usually embedded into the cytoplasm. In cells shifted number of microtubules associated with mature centri- to G0-period (incubated without change of cultured ole and radiating from it. Under the action of these medium for 3 days) cilia were present in 40% (25 of 62) drugs, certain changes of the mature centrioles 144 I.B. Alieva, I.A. Vorobjev / Cell Biology International 28 (2004) 139–150

Fig. 2. Electron micrograph of serial sections showing the ﬁne structure of the centrosome and primary cilium (cross section) in PK cell situated in

G0 period. A–E—active centriole in the region of cilium appearance (sections 1, 3, 4, 5 and 8), S—pericentriolar satellites, A—appendages. F–H—nonactive centriole (sections 1, 5 and 7). Bar=0.2 µm. orientation were revealed by statistical analysis and the and Vorobjev, 1994, 1995). This nonrandom orientation frequency of appearance of different centrosome of the mature centriole correlates with increase in components—pericentriolar satellites, striated rootlets number of pericentriolar satellites on the mature centri- etc. (Alieva et al., 1992; Alieva and Vorobjev, 1995). ole and cytoplasmic microtubules radiating from the Using stereoscopic analysis we did not find any centrosome. Besides active mature centrioles changed change in the three dimensional structure of primary their localization inside the cell—they came between cell cilium in PK cells after all treatment used. But the nucleus and the upper cell surface and were not located axonemes of cilia had appeared after any treatment were at the lateral side from the nucleus as in control cells. much more shorter that in normal cells, and such cilia These changes came along with increase in the per- never came to the cell surface. centage of primary cilia (Fig. 4). Thirty min after As a result of drug treatment mature centriole usually introduction of different inhibitors (A23187, ouabain, became perpendicular to the substrate surface (Alieva DNP, sodium azide), the frequency of primary cilia I.B. Alieva, I.A. Vorobjev / Cell Biology International 28 (2004) 139–150 145

Fig. 3. Electron micrograph showing the fine structure of the centrosome and primary cilium (longitudinal section) in PK cells situated in G0 period. The distal segment of the axoneme of primary cilium is out of the plane of section. The proximal segment of the axoneme is in the membrane vesicle. A—active centriole with the pericentriolar satellite and the cilium and nonactive centriole. B—active and nonactive centrioles with striated rootlet (arrows). Bar=0.5 µm. increased from 60 to 100% compared to the control different centrosome activities separately. Of particular level. Formation of the additional cilia was rather interest was correlation between formation of primary rapid—even 5 min treatment with uncouplers of oxidat- cilia and striated rootlets. ive phosphorylation led to increase in the frequency of Striated rootlets are one of the odd components of primary cilia (Fig. 5). the centrosome in mammalian cells. Striated rootlets were described in different types of non-ciliated cells 3.5. Primary cilia and striated rootlets (Lauiveryns and Boussauw, 1973; Nadezhdina et al., 1979; Sakagushi, 1965; Vorobjev and Chentsov, 1977), Since the action of drugs lead to a number of changes however, they remain largely unexplored. We found that in the centrosome, we analysed the correlation between the number of rootlets in the centrosome is unstable and 146 I.B. Alieva, I.A. Vorobjev / Cell Biology International 28 (2004) 139–150

Table 1 Comparison of immunoﬂuorescent and EM determination of primary cilia formation

Cell line PK REF 3T3 HeLa % ciliated cells, immunostaining (N=100) 28 31 21 2 % ciliated cells, electron microscopy (N=50) 28 32 22 2

Table 2 Formation of the primary cilium in ﬁbroblasts after mitosis (from Alieva et al., 1998)

Cell line % ciliated cells 1–4 h after replating % ciliated intact cells from the same 1234passage REF05–931 3T3 0 7.5 12.5 18 19 Fig. 5. Histogram of frequency of occurrence of the primary cilium in control PK cells (1) and after 5-min treatment of uncouplers of oxidative phosphorylation: DNP (2) and FCCP (3).

no correlation between the appearance of striated rootlets in the centrosome and cilium formation, and conclude that in cultured cells, striated rootlets may have lost their function and are atavistic.

3.6. Primary cilia under the action of microtubule-depolymerizing agents

Primary cilia contain acetylated tubulin in its axoneme, and thus might be expected to be more Fig. 4. Histogram of frequency of occurrence of the primary cilium resistant to microtubule-depolymerizing treatments. (black columns) and striated rootlets (grey columns) in control PK However, we found that in PK cells primary cilia cells (1) and after 30-min treatment of different types of inhibitors: disassemble after cold treatment (2 h at 0 (C), after calcium ionophore A23187 (2); inhibitor of K+/Na+ ATPase ouabain colcemid treatment (1 µg/ml, 4 h) and under high- (3); uncoupler of oxidative phosphorylation DNP (4); plasma mem- pressure treatment (2000 atm, 2 h). In the latter case, brane depolarizer (but not mitohondrial membrane potential) sodium azide (5). however, the cilium did not disappear, but its axoneme was depolymerized while membrane protrusion attached to the distal end of the mature centriole remained (data they run more frequently from mature, but also from not shown). immature (daughter) centriole. In 10% of cells striated It is interesting to note that 45 min after release from rootlets run from both centrioles. Under the action of cold and colcemid treatments, rudimentary primary some metabolic inhibitors, the frequency of association cilia with axonemes of 0.5–1 µm long appeared in the of striated rootlets with immature centrioles increased. majority of cells, while they were present in <10% of Under the action of all metabolic inhibitors used in the control PK cells. This formation of rudimentary cilia present study the frequency of striated rootlets changed. seems to be a non-specific reaction of the centrosome However, it was not correlated with the frequency of related to the burst in the formation of cytoplasmic primary cilia under the same treatment (Fig. 4). microtubules on the centrosome, as happens during According to the function of striated rootlets in basal recovery of microtubule array (Vorobjev and Chentsov, apparatus (“anchors” of cilia and flagella; Hard and 1983). Rieder, 1983), the regulation of cilia and flagella motility Summarizing the results obtained we conclude that (Hyams and Borisy, 1975; Salisbury and Floyd, 1978) formation of the primary cilium after mitosis usually and signal-transforming function (Kleve and Klark, takes several hours, and they never appear in 100% of 1980), the possibility is that those of primary cilia serve interphase cells. However, under experimental treat- the same functions as centrosomal striated rootlets, for ments formation of additional cilia in the interphase example, the anchorage of primary cilia. But we found cells is more rapid process and might take 5–30 min. I.B. Alieva, I.A. Vorobjev / Cell Biology International 28 (2004) 139–150 147

4. Discussion cells. We assume that the primary cilia could keep 4.1. Three hypotheses for the primary cilium function potential to perform sensory functions in cultured cells—at least in some part of cell cultures that descend Since the discovery of primary cilium in 1898 from renal tissue, such as PK cells. (Zimmerman, 1898) three major hypotheses for their function have been put forth. The first is that primary 4.2. The primary cilium is a sensory part of centrosomal cilia are vestigial organelles inherited from an ancestor complex in tissue cells but it is “sleeping beauty” in cells whose cells had motile cilia, and now are of no purpose cultured in vitro in multicellular organisms (Sorokin, 1962). The second is that they are involved in control of the cell cycle Analyzing primary culture of renal epithelial cells we (Tucker and Pardee, 1979). And the third is that primary show that after explantation, when tissue looses its 3D cilia are sensory organelles (Barnes, 1961; Munger, 1958; organization (probably, with some of special tissue Poole et al., 1985; Wheatley et al., 1996). functions) and cultural medium provides constancy of There has been virtually no experimental evidence in surroundings, and cells round up primary cilium dis- support of any of these hypotheses, but the first one, appears. Later on when cells spread on the substrate atavistic hypothesis, is out of interest now. The impor- primary cilium appears in some of them often being very tance of primary cilium at least in vertebrate visual and short with axoneme inside the cytoplasm and located olfactory system is now evident (Brailov et al., 2000; near the cell nucleus. Meanwhile the substantial length Handel et al., 1999; Pazour et al., 2002b; Pazour and of primary cilium as far as the cilia rigidity and bending Witman, 2003; Poole et al., 1985; Praetorius and Spring, are the central and main for cilia functions. In mammals, 2001; Roth et al., 1988; Schwartz et al., 1997; Somlo and the cilia are typically about 5 microns long, but have Ehrlich, 2001) and diseases based on the defects of been observed to be as long as 30 microns on cells from primary cilium, for example polycystic kidney disease kangaroo rat kidney (Wheatley and Bawser, 2000). The (PKD) were described (Hughes et al., 1995; Mochizuki reversible, large-angle bending of the primary cilium et al., 1996; Nauli et al., 2003; Ong and Wheatley, 2003; upon exposure to fluid shear force was experimentally Pazour et al., 2002; Somlo and Ehrlich, 2001; Yoder characterized in vitro, the cilium was then mathemati- et al., 2002). cally modeled as a cantilevered beam and the flexural The first experimental evidence that primary cilia rigidity of the primary cilium was calculated (Schwartz have a function came from studies of a mutant mouse, et al., 1997). It was shown that the removal of primary which has an insertion mutation in the Tg737 gene and, cilium abolished flow sensing (Praetorius and Spring, as a result, develops PKD (Moyer et al., 1994) because it 2001) and bending the primary cilium opens Ca2+ sensi- was unable to assemble normal primary cilia (Pazour tive intermediate-conductance K+ channels (Praetorius et al., 2000). Later it was shown that the mouse Tg737 et al., 2003). protein is homologous to the intraflagellar transport Primary cilium is generally recognized as a sensor protein IFT88 (Pazour et al., 2000), which is involved in with specific receptors receiving external signals (Brailov the bidirectional movement of particles along ciliary and et al., 2000; Handel et al., 1999; Pazour et al., 2000; flagellar microtubules and is essential for ciliary and Pazour et al., 2003) and we suggest that it transduces flagellar assembly (Pazour et al., 2000). them through the centrosome on to radial microtubule According to the second hypothesis primary cilia are system and enhance microtubule related transport be- involved in control of the cell cycle (Ho and Tucker, tween Golgi zone and other cytoplasmic components. 1989; Tucker and Pardee, 1979). There is indirect evi- For normal function primary cilium requires continuous dence that primary cilia abandon liver epithelial cells in external signalling. Treatment of cultured cells with ff G1 and G0 periods may play some role in the inhibition di erent inhibitors mimics such signalling and thus of cell proliferation due to external signalling (Richards stimulates formation of the primary cilia. However, in et al., 1997). However, this possibility remains largely the absence of subsequent signals these cilia do not unexplored. maturate. During the last years, a growing body of evidence It seems likely that formation of the primary cilium is provided strong support for the hypotheses that primary related to the cell–cell contacts—in the cells without cilia have sensory function–sensory transduction (Roth junctions cilium seems to be absent, or at least appear et al., 1988; Schwartz et al., 1997) or mechanoreceptors at the very low frequency. Summarizing, the primary (Praetorius and Spring, 2001; Whitfield, 2003) and cilium, being the sensory part of centrosomal complex in mechanosensors (Schwartz et al., 1997). They were tissue cells appears to be “sleeping beauty” (quiescent) in shown to carry on specific receptors and relaying signals some cultured cells. from their receptors to the cell (reviewed by Pazour and Our data support the idea that foundation of primary Witman, 2003). However, in all these studies the inves- cilium and its maturation are different processes. We tigators dealt with normal tissue rather then cultured suggest that in cultured cells primary cilium cannot 148 I.B. Alieva, I.A. Vorobjev / Cell Biology International 28 (2004) 139–150

Cell surface

1 2 3 45 6

Fig. 6. Scheme of primary cilium formation. Stage 1. Changes in the spatial organization of appendages on the active centriole—they become strictly symmetric. Stage 2. Membrane vesicle attaches to the mature centriole. Stage 3. Initiation of axoneme formation. Stage 4. Elongation of the primary cilium. Stage 5. Outcoming of the primary cilium to the cell surface. Stage 6. Maturation of the primary cilium (process depends on cell differentiation). complete its maturation and function in a regular way micron) and often does not appear at the cell surface. because of the loss of cell differentiation that occurs in Formation of the primary cilium is not associated with homogeneous culture medium with low fluid current. repositioning of the centrosome that occurs in some Very often primary cilium is inside the cytoplasmic types of epithelial cells in situ. Elongation of primary vesicle and does not come to the cell surface. However, cilium occurs in epithelial cells that move out of the cell inhibitory analysis demonstrates that formation of such cycle, but does not happen in resting fibroblasts. rudimentary cilia is a subject for external regulation. Formation of the primary cilium does not inhibit Taking together our observations we suggest that other activities of the centrosome—maternal centriole formation of primary cilium is a multi-step process. with and without cilium has satellites from which cytoplasmic microtubules emerge. In cultured epithelial cells 4.3. Formation of the primary cilium that are out of the cell cycle primary cilium becomes rather long and could be determined using light micro- Use of serial sectioning allowed us to determine initial scope. It is usually located on the upper surface of the steps of the formation of primary cilium. In cultured cell, meanwhile always near the cell nucleus. In fibro- cells centrosome is typically located near the nucleus blasts primary cilia are only few microns long and could rather than near the cell surface, however, the mother be found on the upper surface as well as aside of the centriole does not lose its capacity to form primary nucleus or even under it. We conclude that formation of cilium. Initial step of the formation of primary cilium is the primary cilium comes through the following con- association of the distal end of maternal centriole with secutive steps (Fig. 6): initiation, elongation of the concave–convex vesicle about 0.3 microns in diameter axoneme, fusion of the axonemal vesicle with the plasma probably from the Golgi complex. This vesicle is in firm membrane, and final development and specialization of contact with centriolar appendages and then axoneme the distal part of cilium. It is interesting that at the first begins to grow into concave part of it forming a small stage of primary cilium formation it is possible to see bud. Doublet microtubules of the primary cilium grow changes in the spatial organization of appendages on the directly from the distal part of triplets of centriolar active centriole—they become strictly symmetric. microtubules without any intercept. However, micro- In cells cultured in vitro, the last step seems to be tubules of the axoneme always have lower density abolished and the primary cilium may remain incom- compared to the triplet microtubules. The next step is plete, unable to perform its sensory function. The func- elongation of the axoneme that requires growth of the tion of the primary cilium is performed only in membrane vesicle. Primary cilium at this stage looks like completely differentiated cells and not in proliferating a long cone. Later on membrane vesicle surrounding ones. This may explain different frequency of the axoneme fuses with the plasma membrane and primary presence of primary cilia in cultured cells. cilium comes to the cell surface. Often it is localized in It is interesting to compare formation of the primary the pit on the cell surface. cilium and striated rootlets. Both structures seem to In proliferating epithelial and fibroblasts cells (that have no specialized function in cultured cells. In epi- are not in G0) primary cilium is rather short (about 1 thelial cells striated rootlets are formed simultaneously I.B. Alieva, I.A. Vorobjev / Cell Biology International 28 (2004) 139–150 149 with primary cilia. However, in response to different Alieva IB, Vaisberg EA, Nadezhdina ES, Vorobjev IA. Microtubule treatments the frequency of their formation is different. and intermediate filament patterns around the centrosome in More than that, there is no correlation between the interphase cells. In: Kalnins VC, editor. The centrosome: Academic Press, 1992;103–29. presence of striated rootlets and primary cilium in a Barnes BG. Ciliated secretory cell in the pars distals of the mouse particular cell. Besides striated rootlets are occasionally hypophysis. J Ultrastruct Res 1961;5:453–61. formed in blood cells (Vorobjev and Chentsov, 1977) Brailov I, Bancila M, Brisorgueil M, Miquel M, Hamon M, Verge D. where formation of the primary cilium is inhibited. Localization of 5-HT6 receptors at the plasma membrane of Taken together, the above observations bring us to neuronal cilia in the rat brain. Brain Res 2000;872:271–5. Bystrevskaya VB, Lichkun VP, Krushinsky A, Smirnov VN. Centriole the conclusion that centrosome in vertebrate cells inher- modification in human aortic endothelial cells. J Struct Biol 1992; ited a complete set of features from the cell center of 109:1–12. flagellated single-cell ancestors. It is capable of for- Dahl HA. Fine structure of cilia in rat cerebral cortex. Z Zellforsch mation of the radial microtubule array in the interphase Mikrosk Anat. 1963;60:369–86. cell, participates in the organization of spindle poles, Dalen HA. An ultrastructural study of primary cilia, abnormal cilia forms cilium, and striated rootlets. Location of the and ciliary knobs from the ciliated cells of the guinea-pig trachea. Cell Tissue Res 1981;220:685–97. centrosome close to the cell nucleus and to the Golgi Fonte VG, Searls RL, Hilfer SR. The relationship of cilia with cell complex emphasizes its central role in the spatial organ- division and differentiation. J Cell Biol 1971;49:226–9. ization of cytoplasm. However, numerous functions of Handel MS, Schulz A, Stanarius M, Schreff M, Erdtmann-Vourliotis the centrosome in multicellular organism seems to be H, Schmidt G et al. Selective targeting of somatostatin receptor 3 down regulated. Some attributes of the original cell to neuronal cilia. Neuroscience 1999;89:909–26. center are kept permanently, while others appear only Hard R, Rieder CL. Muciliary transport in newt lungs: the ultrastructure of the ciliary apparatus in isolated epithelial sheets and in from time to time and/or function only in specialized functional triton-extracted models. Tissue Cell 1983;15:227–43. cells. Ho PT, Tucker RW. Centriole ciliation and cell cycle variability during G1 phase of BALB/c 3T3 cells. J Cell Physiol 1989;139:398–406. Hughes J, Ward CJ, Peral B, Aspinwall R, Clark K, San Millan JL et al. The polycystic kidney disease 1 (PKD1) gene encodes a novel Acknowledgements protein with multiple cell recognition domains. Nat Genet 1995; 10:151–60. We wish to thank Dr Bodo Lang and Prof. Keith Hyams JS, Borisy GG. Flagellar coordination in Chlamydomonas Gull for monoclonal antibodies C3B9. We thank V.V. reinhardtii: isolation and reactivation of the flagellar apparatus. Krugliakov for long-term technical assistance in electron Science 1975;12:891–3. Jensen CG, Davison EA, Bowser SS, Rieder CL. Primary cilia cycle in microscopy. Our special thanks to K.M. Smurova for PtK1 cells: effects of colcemid and taxol on cilia formation and excellent assistance in preparation of photographs for resorption. Cell Motil Cytoskeleton 1987;7:187–97. this publication. This work was supported by Grants Jurand A. Some aspects of the development of the notochord in mouse 02-04-48839 for I.A.V and 03-04-48035 for I.B.A. from embryos. J Embryol Exp Morphol 1974;32:1–33. the Russian Basic Science Foundation and by the Grant Kleve MG, Klark WH Jr. Association of actin with sperm centrioles: of President of Russian Federation MD-244.2003.04 for isolation of centriolar complexes and immunofluorescent localization of actin. J Cell Biol 1980;86:87–95. I.B.A. Komarova YuA, Ryabov EA, Alieva IB, Uzbekov RE, Uzbekova SV, Vorobjev IA. Polyclonal antibodies against human -tubulin stain centrioles in mammalian cells from different tissues. Membr Cell References Biol 1996;10:502–12. Latta H, Maunsbach AB, Madden SC. Cilia in different segments of Albrecht-Buehler G. Phago-kinetic tracks of 3T3 cells: parallels be- the rat nephron. J Biophys Biochem Cytol. 1961;11:248–52. tween the orientation of track segments and of cellular structures Lauiveryns JM, Boussauw L. Striated filamentous bundles associated which contain actin or tubulin. Cell 1977;12:333–9. with centrioles in pulmonary limphatic endothelial cells. J Ultra- Albrecht-Buehler G. Function and formation of centrioles and basal struct Res 1973;42:25–8. bodies. In: Kalnins VC, editor. The centrosome: Academic Press, Mochizuki T, Wu G, Hayashi T, Xenophontos SL, Veldhuisen B, 1992;69–102. Saris JJ et al. PKD2, a gene for polycystic kidney disease that Alieva IB, Vorobjev IA. The morphofunctional characteristics of encodes an integral membrane protein. Science 1996;272:1339–42. striated rootlets within centrosomes of the cultured PK-cells. Moyer JH, Lee-Tischler MJ, Kwon H-Y, Schrick JJ, Avner ED, Tsitologia 1989;31:1016–9 (in Russian). Sweeney WE et al. Candidate gene associated with a mutation Alieva IB, Vorobjev IA. Centrosome behavior under the action of causing recessive polycystic kidney disease in mice. Science 1994; uncoupler and the effect of disruption of cytoskeleton elements on 264:1329–33. the uncoupler-induced alterations of the centrosome. J Struct Biol Munger BL. A light and electron microscopic study of cellular 1994;113:217–24. differentiation in the pancreatic islets of the mouse. Am J Anat Alieva IB, Vorobjev IA. Centrosome behavior and orientation of 1958;103:275–311. centrioles under the action of energy transfer inhibitors. Cell Biol Nadezhdina ES, Fais D, Chentsov YuS. On the association of Int 1995;19:103–12. centrioles with the interphase nucleus. J Cell Biol 1979;19:109–15. Alieva IB, Gorgidze LA, Komarova YuA, Chernobelskaya OA, Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X et al. Vorobjev IA. Experimental model for studying the primary cilia in Polycystins 1 and 2 mediate mechanosensation in the primary tissue cultured cells. Membr Cell Biol 1999;12:895–905. cilium of kidney cells. Nat Genet 2003;33:129–37. 150 I.B. Alieva, I.A. Vorobjev / Cell Biology International 28 (2004) 139–150

Ong AC, Wheatley DN. Polycystic kidney disease—the ciliary Sorokin S. Centrioles and the formation of rudimentary cilia by connection. Lancet 2003;361:774–6. fibroblasts and smooth muscle cells. J Cell Biol 1962;15:363–77. Pazour GJ, Dickert BL, Vucica Y, Seeley ES, Rosenbaum JL, Witman Tucker JB, Paton CC, Richardson GP, Mogensen MM, Russel IJ. A GB. Chlamydomonas IFT88 and its mouse homologue, polycystic cell surface-associated centrosomal layer of microtubule- kidney disease gene Tg737, are required for assembly of cilia and organizing material in the inner pillar cell of the mouse cochlea. flagella. J Cell Biol 2000;151:709–18. J Cell Sci 1992;102:215–26. Pazour GJ, San Agustin J, Follit JA, Rosenbaum JL, Witman GB. Tucker RW, Pardee AB, Fujiwara K. Centriole ciliation is related to Polycystin-2 localizes to kidney cilia and the ciliary level is elevated quiescence and DNA synthesis in 3T3 cells. Cell 1979a;17:527–35. in ORPK mice with polycystic kidney disease. Curr Biol 2002a; Tucker RW, Scher CD, Stiles CD. Centriole deciliation associated 12:R378–80. with the early response of 3T3 cells to growth factors but not to Pazour GJ, Witman GB. The vertebrate primary cilium is a sensory SV40. Cell 1979b;18:1065–72. organelle. Curr Opin Cell Biol 2003;15:105–10. Vorobjev IA, Chentsov YS. The dynamics of reconstitution of micro- Pazour GJ, Baker SA, Deane JA, Cole DG, Dickert BL, Rosenbaum tubules around the cell center after cooling. Eur J Cell Biol 1983; JL et al. The intraflagellar transport protein, IFT88, is essential for 30:149–53. vertebrate photoreceptor assembly and maintenance. J Cell Biol Vorobjev IA, Chentsov YS. The ultrastructure of centriole in mam- 2002b;157:103–13. malian tissue culture cells. Cell Biol Int Rep 1980;4:1037–44. Poole CA, Flint MH, Beaumont BW. Analysis of the morphology and Vorobjev IA, Chentsov YS. The ultrastructure of the centrosome in function of the primary cilia in connective tissues: a cellular axolotl hemopoetic tissue. Tsitologia 1977;19:588–603 (in Russian). cybernetic probe. Cell Motil Cytoskeleton 1985;5:175–93. Vorobjev IA, Chentsov YuS. Centrioles in the cell cycle. I. Epithelial cells. J Cell Biol 1982;93:938–49. Poole CA, Jensen CG, Snyder JA, Gray CG, Hermanutz VL, Wheatley DN, Feilen E, Yin Z, Wheatley SP. Primary cilia in cultured Wheatley DN. Confocal analysis of primary cilia structure and mammalian cells: detection with an antibody against detyrosinated colocalization with the Golgi apparatus in chondrocytes and aortic a-tubulin (ID5) and by electron microscopy. J Submicrosc Cytol smooth muscle cells. Cell Biol Int 1997;21:483–94. Pathol 1994;26:91–102. Praetorius HA, Frociaer J, Nielson S, Spring KR. Bending the primary Wheatley DN. Primary cilia in normal and pathological tissues. cilium opens Ca2+-sensitive intermediate-conductance K+ chan- Pathobiology 1995;63:222–38. nels in MDCK cells. J Membr Bio 2003;191(3):193–200. Wheatley DN. Centrioles: the central enigma of cell biology. New Praetorius HA, Spring KR. The renal cell primary cilium functions as York: Premium Press, 1982. a flow sensor. Curr Opin Nephrol Hypertens 2003;12(5):517–20. Wheatley DN. Cilia in cell-cultured fibroblasts. 1. On their occur- Richards WG, Yoder BK, Isfort RJ, Detilleux PG, Foster C, Neilsen rence and relative frequencies in primary and established cell lines. N et al. Isolation and characterization of liver epithelial cell lines J Anat 1969;105:351–62. from wild-type and mutant TgN737Rpw mice. Am J Pathol 1997; Wheatley DN, Wang AM, Strugnell GE. Expression of primary cilia 150:1189–97. in mammalian cells. Cell Biol Int 1996;20:73–81. Rieder CL, Jensen CG, Jensen LCW. The resorption of primary cilia Wheatley DN. Cilia in cell-cultured fibroblasts. III. Relationship during mitosis in a vertebrate (PtK1) cells line. J Ultrastruct Res between mitotic activity and cilium frequency in mouse 3T6 1979;68:173–85. fibroblasts. J Anat 1971;110:367–82. Roth KE, Rieder CL, Bowser SS. Flexible substrate technique for Wheatley DN, Bawser SS. Length control of primary cilia: analysis of viewing cells from the side: some in vivo properties of primary monociliate and multiciliate PtK1 cells. Biol Cell 2000;92:573–82. (9+0) cilia in cultured kidney epithelia. J Cell Sci 1988;89:457–68. Whitfield JF. Primary cilium—is it an osteocyte‘s strain-sensing Sakagushi H. Pericentriolar filamentous bodies. J Ultrastruct Res flowmeter? J. Cell Biochem 2003;15:233–37. 1965;12:13–21. Yoder BK, Hou X, Guay-Woodford LM. The polycystic kidney Schwartz EA, Leonard ML, Bizios R, Bowser SS. Analysis and disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are modeling of the primary cilium bending response to fluid shear. co-localized in renal cilia. J Am Soc Nephrol 2002;13:2508–16. Am J Physiol 1997;272:132–8. Zimmerman KW. Beitra¨ge zur Kenntniss einiger dru¨sen und epithelien Somlo S, Ehrlich B. Human disease: calcium signaling in polycystic [English translation: Contributions to knowledge of some glands kidney disease. Curr Biol 2001;11:R356–60. and epithelium]. Arch Mikr Anat 1898;52:552–706.