Immunodetection of annexins 1 and 2 in ciliated cells from quail oviduct*

Biol Cell (1992) 75, 45-54 © Elsevier, Paris

45

Original article

Immunodetection of annexins I and 2 in ciliated cells from quail oviduct*

Bernadette Chailley ~, Louise-Anne Pradel 2

Centre de Biologie Cellulaire CNRS, 67, rue Maurice Giinsbourg, 94205 Ivry-sur-Seine Cedex; 2 Laboratoire de Biophysique, lnstitut de Biologie Physico-chimique, 13, rue Pierre et Marie Curie, 75231 Paris Cedex, France

(Received 15 February 1992; accepted 10 March 1992)

S u m m a r y - Annexins 1 and 2 are Ca2+-binding proteins related to the cytoskeletal proteins which have been reported to bind in a calcium-dependent manner to F-actin and phospholipids in vitro. Proteins immunologically related to the brain 37-kDa annexin 1 and 36-kDa annexin 2 were characterized by immunoblotting epithelial ciliated cells from quail oviduct. They were detected by immunofluorescence in ciliated as well as glandular cells, using antisera and purified antibodies directed against pig brain annexins. The pattern of labeling was found in the apical part of both cell types, with close membrane association. However, a wider distribution was observed in mature ciliated cells: annexins were localized in the well developed cytoskeletal meshwork in which the ciliary apparatus is tightly anchored. After immunogold labeling, annexins l and 2 were located in the same area as spectrin 240/235 and at the connection sites of F-actin ; both these cytoskeletals proteins were associated with the appendages of the basal body. In contrast, annexins were not detected in immature epithelial cells, while actin and spectrin were present. During ciliogenesis, the staining gradu- ally appeared associated with the lateral and apical membranes. In this cellular model, the annexins may function during exocytosis in gland epithelial cells, where a close cytoskeleton-membrane association is observed; moreover, in ciliated cells, a relationship between cytoskeletal elements of the terminal web and annexins may exist.

annexins / calpactin / Ca 2÷ actin-binding protein / Ca 2÷ phospholipid-binding protein / ciliated cells / epithelium / immunodetection

I n t r o d u c t i o n

Ciliated cells have a highly organized cytoskeleton in their apical part which presumed to play an important role in ciliated cell activity [42]. No precise functions have been clearly demonstrated to date for cytoskeletal elements, but they are involved in basal body orientation and anchorage necessary due to the substantial forces applied during ciliary beat. Apart from the axonemal and basal body sta- ble microtubules, which are the most important cytoskeletal elements constituting cilia [17], labile cytoplasmic microtubules are connected to the basal foot of basal bodies; among them, some microtubules extend parallel to the cell surface in quail oviduct [4] as in mussel gill [39] ; others sink into the apical cytoplasm. Moreover, microtubules f rom peripheral basal bodies are associated with the apical juntional complex in the form of a ring [42]

Most actin microfilaments of metazoan ciliated cells are related to microvilli (which are interspersed between cilia) and their roots which sink into the cortical part of the cell. The actin orientation in microvilli of ciliated cells has been detected after myosin S~ decoration, and the relationship of microfilaments with ciliary structure was previously es- tabl ished. Some microfilaments are closely connected to the basal foot of the basal body [9, 39] or to the upper part of the striated rootlets of cilia [9]. Isolated actin microfilaments also originate from the anchoring sites of

* This paper is dedicated to the memory of Professor Daniel Sandoz. He gave us constant support and encouragement in our study of differentiation and organization of ciliary epithelium.

basal body to the plasma membrane. As in other epithelial cells, an apical belt of microfilaments is associated with the zonula adherens. Peripheral microvilli and basal bodies have actin connections with the belt.

Beneath both the microvillus root and the basal body levels, an apical disk of cytokeratin intermediate filaments extends from desmosomes [24, 44]. The ciliary striated rootlets sink deeply into the bundles of cytokeratin intermediate filaments with which they are closely connected [24, 34]. Among the minor cytoskeletal proteins, actin-binding proteins including myosin [30] and spectrin 240/235 [10] have been found in the apex of ciliated cells. Both are roughly codistributed in the cortical area extending between the apical membrane and the cytokeratin disk, associated with the basal bodies. It is worth noting that in the apical domain of oviduct [10] as well as tracheal [31] ciliated cells, spectrin is not related to the plasma membrane, as is usually found in most of the cells. Spectrin was shown to be associated around the basal foot of basal bodies and the upper part of the striated rootlets [10]. Myo- sin is localized between basal bodies and associated with the apical actin belt [30] in which vinculin has also been observed and characterized (Chailley, unpublished results). Conversely, spectrin has been shown all along the lateral membrane, except in the junctional complex area [10].

To explore anchorage organization in this cell area, it was necessary to determine the proteins which interact with those already described. It appeared of interest to inves- tigate the presence of annexins which belong to a protein family which may mediate cytoskeletal-membrane inter- actions [19, 50]. Annexin 1 (calpactin II), and annexin 2 (calpactin I, lipocortin 2, p36, p90) (see nomenclature in

46 B Chailley, LA Pradel

[14]) which are well charac te r ized b iochemica l ly , are ca l c ium-dependen t p h o s p h o l i p i d - b i n d i n g pro te ins ([21, 22]; review in [13, 29]) and have been repor ted to b ind F-ac t in and spectr in (annexin 2, p90) in vitro in a calc ium- dependent manner [20]. First isolated f rom bird cells, they have been localized in t racel lu lar ly in bo th m a m m a l i a n tissues and in cul ture cell lines [27]. Some i m m u n o f l u o r e s - cence s t u d i e s d e m o n s t r a t e d a n n e x i n l o c a l i z a t i o n unde rnea th the p l a sma m e m b r a n e in f ib rob las t s [25], beneath the microvi l lar a rea in the intestinal epithelial cells [23] and its co loca l iza t ion with spectr in [33]. E lec t ron microscop ic studies con f i rmed the assoc ia t ion with plas- m a m e m b r a n e in f ib roblas t s [46], and in c h r o m a f f i n cells determining the st.ructure o f annexin 2, as fine s trands connecting the two oppos ing membranes o f ch romaf f in granules [36], emphas iz ing the involvement o f annexin 2 in con t ro l o f fusion [16].

In the present s tudy, we detected annexins 1 and 2 in b i rd oviduct c i l ia ted cells by immunof luo rescence , im- m u n o g o l d label ing and i m m u n o b l o t t i n g using prev ious ly charac ter ized an t ibodies directed against pig b ra in annexins [401.

Materials and methods

A n tibodies

Annexin 1 and annexin 2 monomer (p36) were isolated from pig brain (calpactins I and II) as described by Regnouf et al [40]. Purified brain annexins 1 and 2 were subjected to preliminary SDS-PAGE ; the bands excised from gel were homogenized and dialyzed against PBS, then emulsified with Freund's adjuvant before being injected into rabbits. Annexin 2 complex (1990) was isolated from bovine lung as described by Khanna et al [28] and modified by Regnouf et al [40]. 150yg of purified protein emulsified with Freund's adjuvant was directly injected into rabbits. The antibody was tested at a dilution of 1:1000 and reacts only on the p36 heavy chain and not on the pl I subunits. All the antisera were used before and after purification by affinity chro- matography. Antiserum directed against human lymphocyte annexin 2 was a generous gift from Dr Russo-Marie [12].

The secondary antibody was sheep anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC) (Institut Pasteur Production, Paris) or rhodamine for immunofluorescence, with alkaline phosphatase (Promega, USA), for Western blots or with gold GAR G5 (Janssen Laboratory, Beerse, Belgium) for electron immunocytochemistry.

Immuno fl~orescence

Oviduct pieces were removed at three different stages of differentiation : from the immature oviduct ; from the oviduct in which ciliogenesis had been induced in vivo by estrogen ; and from the laying quail oviduct (Coturnix coturnixjaponica). Pieces were quickly frozen with or without prior formaldehyde 2°/0 fixation and sections were obtained with a cryostat. A few frozen sections of unfixed material were also treated by formaldehyde. Then, frozen sections were pre-incubated in 307o bovine serum albumin (BSA) in PBS (phosphate-buffered saline, 150 mM NaCI, 30 mM KCI, 10'mM Na2HPO4, 15 mM KH2PO4, pH 7.4 for 15 min at 20°C, and treated as previously described [8, 10] with antisera or antibodies diluted 1:100 and 1:50 in PBS-BSA for annexins 1 and 2 respectively. After carefully washing in PBS, FITC-labeled antibody diluted 1:100 in PBS was applied for 1 h, after being pre-adsorbed for 30 min over a suspension of ciliated demembranated cells to avoid non-specific binding. For double staining, FITC-labeled phalloidin was used at a dilution of 1:250 for 15 min. Subsequently, the sections were mounted in glycerol- PBS or Mowio1488 (Chimidis, Paris) and observations were made using a Leitz Dialux microscope equipped for UV illumination.

Immunogold labeling

Ciliated cells were isolated from a laying quail oviduct according to the first procedure of Anderson [2] in 20 mM HEPES buffer pH 7.5, 1 mM EGTA, 25 mM KCI, 250 mM saccharose, 0.05070 Triton X-100 and 1 mM PMSF, for 30 min as previously described [8] and the second procedure using a calcium-containing buffer such as 20 mM PIPES buffer pH 5.5, 20 mM CaCI 2, 250 mM saccharose, 0.05070 Triton X-100 with I mM PMSF for 10 min. The suspension of demembranated cells or cortices was cautiously washed in HEPES buffer without Triton, then centrifuged at 2000 g for 30 min in an observation chamber in a monolayer and processed for ultrastructural immunocytochemistry. Isolated cortices on the coverslip were preincubated in PBS containing 3070 BSA, then incubated with antibodies diluted 1:50 and 1:25 for 1 h as above. After several washes in PBS, cortices were incubated for 1 h at 20°C with gold anti-rabbit IgG antibody diluted 1:5 in PBS. After washing, the cortices were fixed with 1 070 glutaraldehyde in 0.1 M sodium cacodylate buffer, post- fixed with 1070 OsO 4, dehydrated and embedded in Araldite. Thin sections were lightly stained with uranyl acetate and lead citrate and examined with a Philips EM300 electron microscope at 80 kV.

Electrophoresis and immunoblotting

Two isolation procedures of ciliated cells were used for biochemical analysis. One consists of isolating cells using Triton X-100 according to [2] ; the other of scraping the surface of the oviduct.

Protein samples from cortices were solubilized in SDS buffer [37] and heated at 100°C for 3 min. One-dimensional SDS-PAGE was carried out according to Laemmli [32] using 1207o linear polyacrylamide slab gel. Proteins were stained with Coomassie brilliant blue R250 (0.25070 w/v) in 5007o methanol, 1007o acetic acid, destalned in 5070 acetic acid and photographed before drying, lm- munoblot analysis was performed by electrophoretically trans- ferring proteins from the unstained SDS-containing gel to 0.10 tzm nitrocellulose filters in Tris-glycine buffer in the presence of 0.1070 SDS [48]. After incubation in 507o non-fat milk in the buffer, the nitrocellulose sheets were incubated with antiserum directed against brain annexins diluted 1:300 in PBS-BSA and antiserum directed against lymphocyte annexin diluted 1:500 for 1 h. Pre-immune serum activity was also controlled. The antigen- antibody complexes were detected with phosphatase alkaline- conjugated IgG (dilution 1:7500) and developed with bromo- chloro-indolyl-phosphate/nitroblue tetrazolium (BCIP/NBT).

Proteins associated with the cytoskeleton from scraped cells were extracted using the method of Zokas and Glenney [50]. After a gentle oviduct scraping, cells were washed twice in PBS in the presence of the protease inhibitors (leupeptin, aprotinin 10 t~g/ml, and 0.1 mM PMSF. The suspension was centrifuged at 500 g for 10 min to obtain a cell pellet I. The supernatant (Sj, washing solutions) was kept and freezed to be analysed. The total proteins of supernatant S~ were controlled before (SO and after a centrifugation for 30 rain at 100000 g collecting a pellet II and a supernatant S 2. The pellet II was submitted to Triton X-100 as the pellet I. The pellet I was freezed at - 8 0 ° C , thawed and resuspended in 200/~1 of 10 mM imidazole buffer, pH 7.3, containing 75 mM KCI, 2 mM MgCI 2, 1 mM NAN3, 0.507o Triton X-100 and protease inhibitors (leupeptin, pepstatin, trypsin in- hibitor, benzamidine and PMSF). The mixture was centrifuged for 60 rain at 28 psi in a Beckmann airfuge and the supernatant fraction was removed. The pellet was suspended in 300 ~1 of 2 mM EDTA, 0.507o SDS, pH 8.5, disrupted using 3.3-s power bursts with a Branson sonifier 220 and incubated for 5 min at 230C. The total proteins in the supernatants and in the fractions of Triton X-100 were estimated by the method of Bradford (1976). For analysis, an EGTA extract (8/~g) of annexins from pig brain was subjected to electrophoresis on SDS polyacrylamide slab gels 1207o with the three samples in order to compare the molecular mass of annexins from quail ciliated cells with those of brain, as well as the reactivity of the purified antibodies. Sepa- rated proteins were electrophoretically transferred to nitrocellulose filter. Blots were probed with an anti-brain p37 and anti-brain p90 (dilution 1:1000). Immunoblots were treated as above.

lmmunodetection of annexins 1 and 2 47

Results

h n m u n o f l u o re scen ce

Using antisera and monospecific antibodies directed against pig brain annexin 1 and annexin 2, no fluorescence was detected in the frozen sections of undifferentiated epithelial cells from fixed or unfixed immature quail oviduct ; the stroma, composed mainly of fibroblasts, was not labeled (fig la). During oviduct differentiation, a hete- rogeneity of labeling is noticed; a delicate staining with anti-annexin 2 (1390) antibodies was observed in the lateral and apical membrane domains of the only luminal cells starting the ciliary differentiation, before cilia were grown. The undifferentiated ceils like the invaginated cells which initiate the formation of tubular glands were not labeled (fig lb-d). Similar apical staining was observed in cilio- genic cells with anti-annexin 1. On sections of laying quail oviduct, the labeling was found throughout the apical border of the ciliated cells at the terminal web level with the p37 and p36 monomer antibodies (figs 2, 3). However, their staining patterns were slightly different according to whether tissues were fixed or unfixed. Anti-annexin 1 appeared to be distributed in the upper part of ciliated cells

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(fig 2a, b), whereas anti-annexin 2 monomer was more peripherally distributed and was present in the lateral domain in unfixed material (fig 3a, b). Cilia were faintly or not stained with those antibodies while the apical part of the cell including cilia was labeled with anti-annexin 2 complex (anti-p90). After fixation, fluorescence appeared at the cilia level with those two antibodies (figs 2c, 3c) and strongly with anti-p90 antibody (fig 4a). Double staining with phalloidin (figs 2d, 3d, 4b) enabled comparison between actin distribution in the cortical part of the ciliated cells including microvilli, and annexin distribution in fixed tissue. Labeling with both anti-annexin antibodies over- lapped the phalloidin staining, but they were clearly not co-distributed. Whether annexin 1 was co-localized with the phalloidin labeling, the annexin 2 detected with anti p90, in addition, extended into the entire length of cilia, and the annexin 2 detected with anti p36 was mainly present in the body of the cell. No staining was observed in mucous cells interspersed between ciliated cells. Triton X-100 treatment in the presence of calcium enabled isolation of demembranated ciliated cells in which labeling was also detected in the terminal web (not shown). Beneath the luminal epithelium, numerous tubular glands responsible for egg white secretion, exhibited weaker labeling closely

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Fig I. lmmunofluorescence detection of annexin 2 in unfixed and fixed frozen sections of quail oviduct before and during its differentiation, a. In immature oviduct, undifferentiated epithelial cells (E) are unlabeled. The stroma (s) rich in fibroblasts is also unlabeled. b-d. In oviduct in ciliogenesis, the undifferentiated epithelial cells like the invaginated cells (short arrows) are unlabeled by anti-annexin 2 (p90). Cytoplasm of cells in ciliogenesis before cilia growing are labeled. A bright labeling is detected at their apical membrane (arrowheads) a faint labeling at their lateral membrane particularly observed on a tangential view of epithelium (b). When cilia were present, they were labeled (long arrow); L, lumen; × 1500.


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Figs 2 - 4 . Immunofluorescence detection of annexins 1 and 2 in epithelial ciliated ceils on unfixed (b) and fixed (e) frozen sections of laying quail oviduct, a. The corresponding phase-contrast b views, d, The corresponding c views double-stained with phaIloidin ; E, epithelium; l, Lumen; × 1 500. 2 b, c. Ant ibody directed against pig brain annexin l (calpactin II). The apical part of ciliated cells is labeled. 3 b, c. Antibody directed against pig brain annexin 2 (calpactin I, p36) ; a staining is detected in the apical and lateral part of the cells. On fixed sections, labeling is more extended. Arrowheads indicate ciliary tips. 4. Immunofluorescence detection of annexin 2 complex (calpactin I, p90) on fixed frozen sections, a. Only the apical part of the ciliated cells is strongly labeled, including cilia, b. The corresponding view double-stained with phalloidin; × 1 500.

Immunodetection of annexins 1 and 2 49

Fig 5. Immunofluorescence detection of annexins on unfixed frozen section of laying quail oviduct. Beneath the epithelium, numerous tubular glands (G) secrete egg white proteins. The apical membrane domains of the secretory cells display thin labeling with anti- annexin 1 and (in inset) anti-annexin 2 antisera; lumen; x 1 500.

associated with the apical plasma membrane when anti- annexin 1 was applied (fig 5). The lateral domain of the secretory cells was also labeled with anti-annexin 2 in unfixed cells (fig 5 - inset), while labeling was not detected after fixation. No fluorescence was shown with non- immune serum either in epithelium or in secretory ceils.

Electron microscopy

Fine localization of antigen-antibody complexes was obtained by pre-embedding immunogold labeling after Tri- ton X-100 permeabilization of ciliated cells in which the apical fibrillar networks and the basal bodies were well preserved. But the treatment used in the presence of calcium to avoid solubilization of annexin induced cell decili- ation and microvillus disorganization. When the apical membrane was preserved, it showed no labeling (figs 6, 8). Gold granules were found in association with thin filaments connected to the basal foot in samples treated with anti-annexin 1 (fig 6) and anti-annexin 2 (fig 7). Labeling was also observed at the upper part of striated rootlets (fig 8) on which granules were superimposed on the peri- odic dark striation when the antibody directed against annexin 1 was used (fig 9). The anchorage fibers present at the distal part of the basal body, enabling its connection to the plasma membrane, were also decorated with the two antibodies (fig 8). When antibodies were applied at a higher concentration, a diffuse pattern of the labeling was detected in the cytokeratin meshwork of the terminal web, in patches. A few of them were distributed on dense struc- tures which may have corresponded to cross or oblique sections of striated rootlets (fig 9). A few gold granules were observed in the cortices with non-immune serum (fig lO).

Protein analysis

Characterization of protein antigens was performed on Western blots from one-dimensional gel electrophoresis. The insoluble protein pattern of ceils isolated by a Triton X-100 treatment (cortices) is shown in figure 1 la and the one of scraped cells in figure 12, ld. The supernatant S~ is rich in ovalbumin 43-kDa and conalbumin 80 kDa secretory proteins elaborated by tubular glands of the oviduct (fig 12, Ib); as observed by light microscopy S 1 is also en- riched in isolated cilia and few cellular fragments.

Depending on the preparative treatment of the cells, the antibodies directed against pig brain annexin 1 recognized a protein with a 37-kDa or a 42-kDa molecular mass. In ciliated cells subjected to a moderate permeabilization by Triton X-100 with caIcium, at a low pH, to be isolated (fig l l b ) the antibody recognized a 42-kDa band plus a weaker band, while it did not do so in those treated with EGTA (fig 11 f). In cells isolated by scraping, then strongly permeabilized in the presence of MgCI 2 at neutral pH, the antibody labeled a 37-kDa band in the Triton-insoluble fraction f rom pellet I (fig 12, 2d) as well as a 37-kDa protein and its proteolytic degradation product 34 kDa in brain extract (fig 12, 2a). No labeling was observed in the Triton-soluble fraction from pellet I (fig 12, 2c). A significant labeling was present in the supernatant S~ (fig 12, 2b) which was due mainly to the presence of numerous cilia since after a I00 000 g centrifugation only pellet II was reactive with the antibodies (fig 13b, c); the Triton-soluble and insoluble extracts from the pellet II were labeled indicating that a soluble pool plus a cytoskeleton pool of annexins 1 and 2 exist in cilia (fig 13).

In contrast, the antibodies directed against pig brain annexin 2 recognized always a 36-kDa band in each cell


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Figs 6 - 1 0 . Pre-embedding immunogold labeling of ciliated cells after Triton X-100 treatment with calcium, x 82 500. 6, 7. Trans- verse thin sections showing basal bodies with their lateral basal foot on which gold particles are associated (arrows) (6: anti-annexin l ; 7 : anti-annexin 2). 8. Annexin 2 (p36) antibody labeling is distributed on filamentous material associated with the upper part of the striated rootlets and the proximal pole of the basal body (arrows). Gold particles are also detected on the anchorage fibers (arrowheads) which link basal bodies to the plasma membrane. Cytokeratin intermediate filaments are unlabeled, but faint, diffuse labeling is noted. 9. Annexin 1 (p37) antibody labeling is distributed on the dark striation of the ciliary rootlets (arrows). Particle patches on dense material are dispersed in the cytokeratin meshwork of the terminal web. 10. Control sample treated with non-immune serum shows non-specific labeling.

lmmunodetect ion of annexins 1 and 2 51

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Fig 11. lmmunodetection on Western blots of proteins from Tri- ton X-100 permeabilized eiliated cells in the presence of calcium (b - e) or in the presence of EGTA ( f - g) and from rat brain syn- aptosomes (h). First lane: standard proteins. Lane a: One dimensional SDS-PAGE 12% stained with Coomassie blue; lanes b, f, h : Western blot treated with anti-brain 37-kDa annexin ! ; lanes c, g: Western blot treated with anti-brain 36-kDa annexin 2; lane d: Western blot treated with anti-lymphocyte annexin 2; lane e: Western blot treated with non-immune serum.

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Fig 12. lmmunodetection on Western blots of Triton X-100 protein extract from scraped ciliated cells (pellet 1). 1: One- dimensional SDS-PAGE 12°70 stained with Coomassie blue. First lane: standard proteins: lane a: brain EGTA-extract; lane b: supernatant St; lane c: Triton X-100 soluble fraction from pellet I; lane d; Triton X-100 insoluble fraction from pellet 1. 2: Western blots treated with anti-brain 37-kDa annexin 1. 3: Western blots treated with anti-brain 36-kDa annexin 2.

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Fig 13. Immunodetection on Western blots of proteins from supernatant S L of scraped cells. Lane a: supernatant $2; lane b: Triton X-100 soluble fraction from pellet 1I; lane c: Triton X-100 insoluble fraction from pellet 11. 1 : Treatment with anti-brain 37-kDa annexin I ; 2: Treatment with anti-brain p90 annexin 2.

p r epa ra t i on , ei ther f rom the permeabi l i zed , i so la ted cells with ca lc ium (fig 1 lc) , or f rom the scraped and then permeabi l ized cells in which the label ing was present main ly in the insoluble f rac t ion (fig 12, 3d). An t i se rum di rec ted against the lymphocy te annexin 2, previous ly cal led l ipocor t in 2, when used in pro te in blot t ing, recognized the 36-kDa band (fig 1 ld) . On the o ther hand , a 6 7 - 6 8 - k D a band was weakly recognized by serum against b ra in annexin 2 in EGTA- t r ea t ed cells (fig 1 lg). No higher molecular mass pro te in was labeled. N o n - i m m u n e se rum was unreact ive on Wes te rn blots o f c i l ia ted cells (fig 1 le) and o f a l ipocor t in -enr iched f rac t ion (not shown). The speci- ficity o f the an t ibod ies is shown in f igure 14; no cross- react iv i ty with o ther annexins was detec ted .

Discussion

In the present repor t , we d e m o n s t r a t e that pro te ins im- munological ly related to bra in annexins are present in quail ov iduc t cells. Both soluble and cytoskele ta l forms o f annexin 1 and annexin 2 (heavy chain p36 and t e t r amer p90) were detected by immunof luo rescence . We have now charac te r ized the de te rgen t - inso lub le fo rm o f annexins in isolated ci l iated cells as previously done in f ibroblas ts [11, 25, 33, 38]. In ci l ia ted cells annexin 2 exists as a 36-kDa pro te in independen t o f the p repa ra t ive t r ea tmen t o f the cells. In contras t , the molecular mass o f annexin 1 appears to depend on the prepara t ion method. Annexin 1 in ciliated cells p repared accord ing to Z o k a s and Glenney [50] appears with an expected 37-kDa molecu la r mass as in pig and rat tissues in which ant i -annexin 1 was specific towards a po lypep t ide o f molecu la r mass 37-kDa [40], whereas in ciliated cells p repared according to Ander son [2] it appears with a 42-kDa molecu la r mass. This change in molecu la r mass has a l ready been observed in rat cu l tu red nervous cells and rat synap tosomes (fig l l h ; unpubl i shed results f rom Pradel) . This molecular mass variabi l i ty could be due to the exper imenta l cond i t ions wherein the t issue extracts


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Fig 14. lmmunoreactivity of antibodies on EGTA-extracts of adrenal medulla (b), spinal cord (c), cortex (d) and cerebellum (e) from pig. Standard proteins (a). 1 : Coomassie brilliant blue staining. 2 : Immunoblotting with anti-brain annexin 1. 3 : With anti-brain annexin 2. 4: With anti-lymphocyte annexin 2 (from [40]).

were submitted. The state of phosphorylation of the protein could also be the responsible factor of the difference of molecular mass. This observation indicates that annexin 1 is sensitive during preparation, and explains that its molecular mass is variable between 35- and 40-kDa in the literature.

The highest levels of annexins 1 and 2 were detected in lung, intestine and placenta which are all organs of ex- change. Although annexins are well characterized, their function remains undefined. However, several hypotheses have been considered, including their possible involvement in secretory processes, especially in exocytosis [1, 5, 16, 36, 451, in organization of cytoskeletal-membrane inter- actions [19, 50] and in cell differentiation [6, 47, 49]. The localization of these proteins in another type of cell may enhance o~r knowledge of their role. Bird oviduct has the advantage of displaying a mucociliary epithelium which covers numerous tubular glands secreting the proteins of the egg white. Thus, by immunofluorescence on frozen sections, it was possible to compare annexin detection and distribution in both ciliated and glandular cells which pos- sess different functions.

Our results with the resolution of the light microscope indicated that annexins 1 and 2 are localized in the apical part of the polarized cells, but appear to have a slightly different pattern according to the cell type. Firstly, in tubular gland cells which do not display a terminal web, annexins 1 and 2 are closely apposed to the apical plasma membrane, as expected. In ciliated cells, which display an exceptionally developed apical cytoskeleton [42], annexins 1 and 2 are present with a high level of staining in the whole apical domain including cilia. However, the be- havior of the proteins depending on treatment suggests that a soluble pool which can be stabilized by fixation may exist in the apical part of the cells and especially in cilia. The

major element of this pool appears to be annexin 1. Across the ciliary membrane, calcium movements occur which could involve calcium binding proteins. Annexins 1 and 2 might contribute in intracellular signal pathways because of their ability to reversibly associate with membrane phos- pholipicls in a calcium-dependent manner and to be phosphorylated by protein kinases. Annexin 2 also occurs over the lateral plasma membrane, with which it appears to be strongly bound and in a insoluble form, since it is observed in fixed and unfixed cells. Its submembrane localization in epithelial cells can be compared with that observed in fibroblasts and cultured cells.

By contrast, detection of annexins 1 and especially 2 in the detergent-insoluble phase using a biochemical ap- proach, and at the cortical level of the mature ciliated cells using immunofluorescence, suggests that both annexins are also tightly bound to cytoskeleton.

In the apical part of the ciliated cells, annexins 1 and 2 may be associated with microfilaments organized in the area [9]. In in vitro experiments, annexins 1 and 2, in contrast to other annexins, were demonstrated to link F-actin [22]. Double staining with phalloidin shows that annexins are overlapping microvilli and cilia in which actin has been previously localized and characterized [8, 43] ; the amount of annexin 2 complex could be higher than annexin 1 in this domain. Our observations are in agreement with re- cent results obtained on epithelial m a m m a r y cells showing that annexin 2 is located in microvilli [26].

Annexin 2 could also be associated with the presence of spectrin in the apical and lateral sites o f ciliated cells. CoIocalization of these two proteins was recently observed in the cuticular plate of rat hair cells with the same anti- annexins [15]. Moreover, the connecting sites of the arrow ends of microfilaments [9], upon which spectrin was previously detected, are also colocalization sites of annexin

Immunodetection of annexins 1 and 2 53

2. However, annexin 2 distribution is wider than that of spectrin, which is absent from cilia and microvilli, as previously shown [10, 31]. The association of annexin 2 and spectrin is supported by the fact that annexin expression is correlated with spectrin expression; indeed, both proteins are absent f rom the cell's apical domain throughout early development of the oviduct. Spectrin is absent until the terminal web develops [10], while actin is already present in microvilli at the cell surface of undifferentiated cells [7]. Our observations are in agreement with the hypothesis of colocalization of spectrin and annexin 2 in other cells [22, 33].

In electron microscopy immunogold labeling did not reveal a characteristic association of these annexins with spectrin. Annexins present in the terminal web appear with a scattered distribution. This might have been due to a relo- cation of the proteins during the isolation of the cells. Although the possibility should not be ruled out, annexins could be associated with the cytokeratin meshwork as it appeared in enterocytes [23]. Such an association with intermediate filaments was shown with neurofilaments for another protein of the annexin family [18]. Annexins may play a role in binding between striated rootlets and the cytokeratin meshwork, or could mediate the events tak- ing place at this level of ciliary apparatus knowing Ca 2÷- ATPase activity has been detected in the striated rootlets by cytochemical studies [3, 41], but until now no data have supported this observation.

The involvement of annexins in cell growth and differentiation has been postulated indirectly; indeed, annexin 2 expression increases in the embryonic rat central nervous system [35] and in cultures of rat pheochromocytoma PC-12 cells which underwent terminal differentiation in response to nerve growth factor [47]. The appearance of annexin 2 only durifig ciliary differentiation might later be implicated in specific functions of the differentiated cell; it could act as submembrane cytoskeletal elements in the lateral and apical membranes. It could also partici- pate in events involving the ciliary apparatus which is anchored in the terminal web. In the immature oviduct, s t roma fibroblasts do not express annexins, while fibroblasts in culture do [33], reinforcing the idea that the expression of this protein is regulated independently according to cellular function or cell type.

Acknowledgments

We gratefully thank L Ovtracht for her helpful advice and stimulating discussions ; S Busson for her interest and encourage- ments of this work and critically reading of the manuscript ; B Rothhut and F Russo-Marie for generously providing antiserum to human lipocortin ; J Rutin for his skillful technical assistance in electron microscopy and M Louette for the photographic work.

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Immunodetection of annexins 1 and 2 in ciliated cells from quail oviduct*