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

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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). 1065-6995/04/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2003.11.013 140 I.B. Alieva, I.A. Vorobjev / Cell Biology International 28 (2004) 139–150 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.
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