immunocytochemical study of the formation of striated ...(1975). these solutions were sonicated at...

Immunocytochemical study of the formation of striated rootlets during

ciliogenesis in quail oviduct

MICHEL LEMULLOIS* and MARIE-CHANTAL MARTY

Centre de Biologic Cellulaire C.N.R.S., 67 me Maurice Giinsbourg, 94205 Ivry sur Seine cedex, France

•Present address: Laboratoire de Biochimie des Transports cellulaires, C.N.R.S., U.R.A. D 1116, Bat 432, Universite Paris Sud, 91405 Orsaycedex, France

Summary

In quail oviduct, a 175K (K=103Mr) protein associ-ated with striated rootlets was previously identifiedby Klotz and co-workers using monoclonal anti-body CC310. As this monoclonal antibody recog-nizes several proteins on immunoblots of ciliatedcells, we prepared a polyclonal antibody mono-specific to the 175K protein by intrasplenic immu-nization of mice. Immunofluorescence study con-firmed the distribution of the 175K protein at theapical part of the ciliated cell and its absence inother epithelial cells. Immunogold staining showedthat this protein was strongly associated with thefibrillar axis of striated rootlets. The absence oflabeling on striation suggested that rootlets werecomposed of several proteins, with one group form-

ing the fibrillar axis and the second forming thestriation. The formation of striated rootlets duringciliogenesis was studied using this polyclonal anti-body. The 175K protein appeared at the beginningof centriologenesis in fibrillar material locatedaround dense granules, and then around the gener-ative complex. The formation of rootlets began atthe basal pole of migrating basal bodies. Theelongation of the rootlet axes took place when basalbodies were anchored to the plasma membrane.

Key words: striated rootlets, ciliogenesis, intrasplenicimmunization, immunocytochemistry, quail oviduct.

Introduction

The apical pole of ciliated cells is characterized by theciliary apparatus and a highly polarized and developedcytoskeleton (for review, see Sandoz et al. 1988). Theciliary apparatus is composed of about 200 basal bodies,which act as nucleating structures for axonemal as-sembly. Different appendices are associated with basalbodies, including alar sheets, basal feet and striatedrootlets. Among these appendices, striated rootlets seemto be responsible for the association of basal bodies withcytoskeleton, since numerous contacts between rootletsand cytoskeleton components have been documented. Inmacrocilia cells of the Ctenophore Beroe, a large actinfilament bundle is associated with striated rootlets inmature cells (Tamm and Tamm, 1987) as well as inciliogenic cells (Tamm and Tamm, 1988). In quailoviduct, actin is associated with the apical part of striatedrootlets (Chailley et al. 1989), but these striated rootletsare also associated, in their basal part, with intermediatefilaments (Sandoz et al. 1983), which have been charac-terized as cytokeratin filaments (Gounon et al. 1987;Lemullois et al. 1987a).

The composition of the ciliary apparatus still remainslargely unknown. The striated rootlets are composed offibrillar material transected by plates of dense material

Journal of Cell Science 95, 423-432 (1990)Printed in Great Britain (C) The Company of Biologists Limited 1990

(Anderson, 1972). Various earlier studies have shown awide range of composition in the different species studied(see discussion).

The genesis of striated rootlets is also unknown. Threeprocesses have been described in the literature: (1) inchicken trachea, according to Kalnins and Porter (1969),the rootlets appear in a specific part of the cytoplasm andthen hang onto basal bodies after anchoring to the apicalmembrane. (2) According to Anderson and Brenner(1971), in monkey oviduct striated rootlets begin to formon the anchored basal bodies. The components of rootletscome from dense granules that appear under the basalbodies and disappear in mature ciliated cells. (3) In theCtenophore Beroe (Tamm and Tamm, 1988), basalbodies are formed near the Golgi apparatus and striatedrootlets develop from the proximal end of basal bodiesprior to the migration phase.

In order to explain rootlet formation during ciliogen-esis in quail oviduct, we used an antibody that specificallyrecognizes a protein associated with striated rootlets.Recently, Klotz et al. (1986), using monoclonal antibodyCC310, identified two major proteins of 40 and 175K(K=103Mr) in ciliated cells of quail oviduct. The 175Kprotein was characterized as a striated-rootlet-associatedprotein.

As this monoclonal antibody recognizes several pro-

423

teins in ciliated cells, it cannot be used for studyingrootlet formation. Thus, we produced a polyclonal anti-body raised against the 175K protein by intrasplenicimmunization. Using monoclonal antibody CC310, weidentified the 175K protein on immunoblots of basalbody components; the 175K protein was isolated anddeposited in the mouse spleen. With this antibody, wefocused our study on the distribution of the 175K proteinin striated rootlets and its role in rootlet formation duringciliogenesis in quail oviduct.

Materials and methods

Isolation of corticesCiliary cortices from laying quails and ciliogenic cortices fromimmature quails, which received three daily intramuscularinjections of 20 /.ig of estradiol benzoate in 0.1ml peanut oil,were prepared according to the first procedure described byAnderson (1974): oviduct segments were filled with 20 mMHepes buffer, pH7.5, 1 mM EDTA, 25 mM KC1, 250 mMsucrose, 1 mM phenylmethane sulphonyl fluoride (PMSF) and0.05 % Triton X-100. The segments were clamped at both ends,placed in 20mM Hepes buffer, pH7.5, 1 mM EDTA, 25mMKC1, and stirred for 2min on a vortex mixer every 5 min for30min at 4°C. The segment was collected, washed twice withmedium without Triton X-100 and sucrose.

Gel electrophoresisProtein samples were prepared from ciliary cortices by dissolv-ing a pellet of these structures in SDS buffer according toLaemmli (1970) or urea-buffer A according to O'Farrell(1975). These solutions were sonicated at 40W for 1 min by 1-spulses and the SDS-containing sample was then heated at100°C for 5 min. The one-dimensional SDS-PAGE was per-formed according to Laemmli (1970), with the modificationintroduced by Porzio and Pearson (1977). A 5 % to 15 % lineargradient of acrylamide was used. Gels were stained withCoomassie Blue R-250 (0.25% (w/v)) in 50% methanol, 10%acetic acid. The two-dimensional IEF/SDS-PAGE was per-formed according to O'Farrell (1975). Isoelectrofocusing (IEF)gel was made with 2% ampholines, pH range 3.5-10. Thedetermination of the pH gradient was measured with a contact-radiometer. The polyacrylamide gel slab chosen for seconddimension contained 8% acrylamide. The proteins were col-ored with AgNC>3 according to Morissey (1981).

ImmunoblotsProteins from unstained polyacrylamide gel slabs were trans-ferred onto nitrocellulose niters (Schleicher and Schuell,0.45 ,um) by simple diffusion, according to Bowen et al. (1980).

The nitrocellulose filter was then incubated in 3 % NP-40 inTris-buffered saline (TBS) for 30 min at room temperature andthen in 5 % skimmed milk or 3 % bovine serum albumin (BSA)in TBS at 37°C for 1 h. Monoclonal antibody CC310 (undilutedculture supernatant) or polyclonal antiserum diluted 1/250 inTBS was applied to a nitrocellulose filter in plastic bags at 4°Covernight. The filters were washed with three changes ofTBS-Tween over a 30-min period, then incubated for2 h 30 min with the second labeled antibody diluted in 1/100TBS, and the filter was washed three times in TBS.

For monoclonal antibody CC310, we used a goat anti-mouseIg coupled with radioactive 12SI (Amersham Corp., ArlingtonHeights, IL). The development of this antibody was carried outby exposing the filter to contact with Kodak XAR film with aDupont Cronex lightening-plus AC screen, for 24 h at —70°C.

For the polyclonal antibody, the second antibody was a goatanti-mouse Ig coupled to horseradish peroxidase (InstitutPasteur Production, Paris, France). Peroxidase activity wasrevealed using diaminobenzidine (DAB) and H2O2.

Preparation of immunogenProtein samples from laying quail were prepared and separatedon polyacrylamide gel slabs as described above. A portion ofunstained polyacrylamide gradient was transferred onto anitrocellulose filter. An immunoblot using monoclonal antibodyCC310 enabled localization of the 175K protein on nitrocellu-lose paper and on a stained polyacrylamide gel slab. The bandof acrylamide gradient containing the 175K protein was cut outwith a razor blade, made up and left in 50 mM NH4HCO3 with0.1 % SDS for one night at 37°C. After centrifugation at 500 g,the supernatant was collected and dialysed against water for 24 hat 4°C, then lyophilized. After lyophilization, the 175K proteinwas removed in 0.1 M PBS at a rate of 1 mg protein per ml ofbuffer.

Immunization of miceIntrasplenic immunization was performed according to Nilssonet al. (1987). Ten two-month-old Balb/c mice were anesthe-tized by intraperitoneal injection of 10 jig of pentobarbital pergram of body weight. After skin and abdominal wall incision,the spleen was exposed. A solution of 10 fi\ of PBS with 10 fig of175K protein was injected into the spleen. The spleen wasreturned to the abdominal cavity and abdominal wall and skinswere sutured separately. This operation was repeated threetimes at 15-day intervals. The pre-immune sera were collected24 h before the first immunization. The serum was collected 15days following the last immunization.

hnmunocytochemistryIndirect immunofluorescence microscopy. Ciliary cortices

centrifuged at 2000g in an observation chamber (Sandoz et al.1982) and 6-mm-thick frozen sections were preincubated withPBS containing 3 % BSA, then incubated for 1 h with undilutedculture supernatant containing monoclonal antibody CC310 orpolyclonal antibody diluted 1/100 in 0 . 1 M PBS and washedthree times in PBS 0.1 M. Fluorescein-conjugated IgG goatanti-mouse Ig (Institut Pasteur Production, Paris, France)diluted l/lOO in PBS was applied for 40min. The frozensections and ciliary cortices were washed three times in PBS,mounted in Mowiol (Mowiol 488, Chimidis, Paris, France) andobserved with a Leitz Dialux microscope equipped for ultra-violet illumination.

Pre-embedding immunogold staining. After incubation withPBS/BSA, isolated ciliary cortices and ciliogenic cortices wereincubated with polyclonal antibody diluted in 1/50 in 0.1 M PBSor with undiluted culture supernatant containing CC310. Thecortices were washed three times in PBS and incubated for 1 hwith gold-labeled goat anti-mouse IgG (GAM G10 EM grade,Janssen Laboratory, Beerse, Belgium) for polyclonal antibodyand with a gold-labeled goat anti-mouse IgM (GAM G5 EMgrade, Janssen Laboratory, Beerse, Belgium) for monoclonalantibody CC310.

Each labeled antibody was diluted 1/5 in PBS. Controlsamples were incubated with the second antibody only. Corticesand ciliogenic cells were fixed in 3 % glutaraldehyde in 0 . 1 MPBS, washed and then postfixed with sodium cacodylate buffercontaining 1 % OsO4, dehydrated in ethanol and flat-embeddedin Araldite. Thin sections stained with uranyl acetate and leadcitrate were examined with a Philips EM 300 electron micro-scope at 80 kV.

Post-embedding immunogold staining. Small pieces of quail

424 M. Lemullois and M.-C. Marty

oviduct were fixed with 3 % paraformaldehyde and 0.5 °Ioglutaraldehyde in 0 . 1 M sodium cacodylate for l h . The fixedtissue was washed in 0.1 M cacodylate buffer and dehydrated ina progressive ethanol series. Then the pieces were embedded inLR White (London Resin, England), which polymerized after24 h at 60 °C without addition of any hardener or accelerator.Thin sections were collected on nickel grids and stored over-night in distilled water at room temperature. Then, the gridswere treated for 1 h with 3 % BSA in 0.1 M PBS and thereafterincubated for 1 h with polyclonal antibody diluted 1/10 in PBS.After several washing in PBS a gold-labeled goat anti-mouseIgG (GAM G10, EM grade, Janssen Laboratory, Beerse,Belgium) diluted l/20 in PBS was applied for 1 h. The gridswere washed in PBS and distilled water. Finally, the treatedsections were stained with uranyl acetate and examined with aPhilips EM 300 electron microscope at 80 kV. For controls, theantiserum was omitted.

Results

Characterization of the polyclonal antibodiesImmunoblots. The typical pattern of protein from

ciliary cortex analysed by one- and two-dimensional gelelectrophoresis are shown, respectively, in Fig. 1A, lanea, and Fig. IB. The immunoblots from one-dimensionalgel electrophoresis (Fig. 1A, lane c) and two-dimensionalgel electrophoresis (Fig. 1C) demonstrated that the im-mune sera used for all immunocytochemical studiesreacted only with a protein of 175K. The pH; of thisprotein is about 6.8. For the six mice that survived afterimmunization, we obtained the same results. Usingmonoclonal antibody CC310, we detected three majorproteins of 40, 62 and 175K and several minor proteins(Fig. 1A, lane b).

Immunofluorescence. On frozen sections of layingquail oviduct and isolated ciliary cortices, we observed,with monoclonal antibody CC310, strong staining in theapical part and slight labeling in the basal part of eachciliated cell (Figs 2A and 3A). Using the polyclonalantibody, we detected strong labeling only in the apicalpart of the ciliated cell (Figs 2B and 3B). Mucus cells andglandular cells were not labeled with these two anti-bodies.

Electron microscopy. After pre- and post-embeddingimmunogold staining using polyclonal antibodies(Fig. 4A,B,C), the striated rootlets were decorated bygold granules. No other ciliary structures such as basalfeet, anchoring fibers or basal bodies were significantlystained. In these ciliated cells, the terminal web wasunlabeled (Fig. 4A and B), in contrast to results obtainedwith CC310 (Fig. 5). With the CC310 antibody, the goldgranules were located on striated rootlets around thebasal body and on dense material associated with thecytokeratin network.

In the same preparation of cortex, striated rootlets aremore or less extracted by incubation with Triton X-100.This difference could be due to a difference in accessi-bility of ciliated cells to detergent, the ciliated cells fromthe top of villi being more accessible that those from thebottom. The intensity of gold labeling obtained withimmune serum by the pre-embedding method (Fig. 4Aand B) was dependent upon the extraction of rootlet

components. When striated rootlets were not extractedwith Triton X-100 (Fig. 4A), the striations were clearlyvisible, particularly in the apical part of the rootlets; onlya few gold granules were associated with them. Whenstriated rootlets were extracted with Triton X-100(Fig. 4B) a fibrillar axis appeared and we observednumerous gold granules located in the axes of rootlets.

In post-embedding immunogold staining (Fig. 4C),the gold granules were distributed alone the rootlets,irrespective of striation. No other structures werestained. Polyclonal antibodies recognized a 175K protein,which was mainly present in the axes of striated rootlets.

Study of rootlet formation with the polyclonal antibodyagainst the 175K proteinIn undifferentiated cortices (Fig. 6), the two centrioles ofdiplosomes were associated with the apical membraneand gave rise to the primary cilia. After pre-embeddingimmunogold staining, the gold granules were located onthin striated rootlets of the proximal centriole as well asaround it.

During centriologenesis, centrioles were generatedwithin each cell according to the two different pathwaysdescribed by Anderson and Brenner (1971). In thecentriole pathways, the diplosome was surrounded bydense material and granules. Procentrioles were formedfrom this material. In the acentriolar pathway, numerousdense granules appeared near the Golgi apparatus andthen merged into large complexes: the deuterosomes. InFig. 7, we observe an early stage of centriologenesis;dense granules and native deuterosome were not decor-ated. Gold granules were located on the striated rootletassociated with the diplosome and decorated filamentousstructures were located between the dense granules.

In the acentriolar pathway, once the deuterosomeswere generated, procentrioles appeared around them. Atthis stage (Fig. 8) gold granules labeled the fibrillarmaterial surrounding nascent centrioles, but the centriolecores and deuterosome cores were not labeled.

During the migration phase (Fig. 9), gold granuleswere located only at the basal pole of the migratingcentrioles. No other structure was labeled. After theanchoring phase, cilia and striated rootlets began to grow(Fig. 10). Nascent striated rootlets were decorated. Nu-merous dense granules appeared just under the basalbodies. These granules were unlabeled, but the fibrillarmaterial that was located between dense granules wasdecorated. No labeling was observed on cilia inelongation, or in other structures in ciliated cells.

Discussion

In the present report, we describe the preparation andcharacterization of a polyclonal antibody that recognizes a175K band associated with striated rootlets in ciliatedcells of quail oviduct. This immunization technique byintrasplenic deposit (Nilsson et al. 1987) enables one toobtain a monospecific polyclonal antibody from a smallquantity of protein separated by electrophoresis.

The antisera obtained from different mice recognized

Formation of striated rootlets 425

200 000_

116 250_97 4 0 0 - ^

66 200_

21500-14400-

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Fig. 1. A. Lane a: one-dimensional SDS-PAGE (5% to 15%) of isolatedciliary cortices stained for proteins with Coomassie Brilliant Blue. Lane b:Western blot of ciliary cortices treated with the monoclonal antibodyCC310. Three major protein bands of 40, 62 and 17SK and several minorprotein bands are observed in the cortex sample. Lane c: Western blot ofciliary cortices treated with polyclonal antibody. One simple band of 175Kis detected in ciliary cortices. Lane d: Western blot of ciliary corticestreated with non-immune serum. The 17SK band is unlabeled. B. Two-dimensional PAGE, separation of ciliary cortex peptides stained with silver.Arrow indicates the 175K protein. C. Immunoblot analysis of ciliarycortices treated with polyclonal antibody, showing the high specificity ofthe antibody.

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116 250— - -

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only one 175K band on immunoblots from Triton X-100-insoluble proteins of quail oviduct ciliated cells. Thissingle band, observed in each blot, demonstrated thatthese immune sera are monospecific, whereas monoclonalantibody CC310 recognized several proteins that prob-ably share a common epitope.

The 175K protein was exclusively located at the apicalpole of ciliated cells as shown by immunofluorescence.Using CC310, we obtained, like Klotz et al. (1986), basal

and apical pole labeling. We confirmed, using polyclonalantibody, that the labeling of the basal pole of ciliatedcells observed using monoclonal antibody CC310 doesnot arise from the detection of the 175K protein.

Immunogold study confirmed that the 175K protein isexclusively associated with striated rootlets and not withother structures. We conclude that the dense materialthat was associated with the cytokeratin network anddecorated the monoclonal antibody . CC310 does not


Figs 2 and 3. Immunofluorescence study of ciliated cells.Fig. 2. A. Frozen section of quail oviduct epithelium treatedwith monoclonal antibody CC310. X2000. The apical part ofciliated cells (arrowhead) is strongly labeled. Their basal part(arrow) shows point staining. Mucus cell (me) and glandularcells (gc) are not labeled. 1, lumen. B. Frozen section of quailepithelium treated with polyclonal antibody. X2000. Theciliated cells show strong staining of their apical part(arrowhead). No other structure in ciliated cells, other cells,mucous cells (me) or glandular cells (gc) is stained. 1, lumen.Fig. 3. A. Isolated cortex treated with the monoclonal

antibody CC310. Staining is associated with the apical part (arrowhead) and the basal part (arrow) of ciliated cortex. X3500.B. Isolated cortex treated with polyclonal antibody. Labeling is only associated with the apical part (arrowhead) of ciliary cortex.X3500. In all cases, the cilia are unlabeled. C. Phase-contrast visualization of the ciliary cortex. X3500.

contain the 175K protein. As for the distribution of 175Kprotein, Klotz et al. (1986), using monoclonal antibodyCC310, could not determine whether the 175K proteinwas a genuine protein of striated rootlets or an associatedprotein.

Using our polyclonal antibody and pre-embeddingimmunodetection in ciliary cortices (Fig. 4A and B) weprovided further information concerning the distributionof the 175K protein. Our observations by pre-embeddingimmunogold staining show that the more striated rootletsare extracted with Triton X-100, the more intense is thestaining. In post-embedding immunogold staining, weobserved staining of rootlets with an absence of period-icity. These results indicate that distribution of the 175Kprotein along the rootlets is independent of striations,and the 175K protein seems to form a fibrillar axis that ispartly hidden from antibodies by the dense striationmaterial. From these results, we conclude that rootletsare composed of at least two groups of proteins, the firstconsisting of proteins like the 175K protein that forms thefibrillar axis, and the second consisting of those causingstriation. This conclusion confirms the earlier hypothesisof Anderson (1972), who described the rootlets as taper-ing bundles of longitudinally arranged fibers embeddedin a periodic light material.

Thin striated rootlets in the diplosomes of immature

epithelial cells are also composed of two groups ofproteins. One of these groups consists of the 175Kprotein and forms the fibrillar axis of the rootlet. Differ-ent components of striated rootlets and flagellar rootshave been described in earlier studies. Amose( al. (1979)isolated a protein of 90K in root fibers of the flagellateTrichomonas. In Naegleria gruberi, Larson and Dingle(1981) detected a 170K protein as a major component offlagellar roots. Marano et al. (1985) observed three majorproteins of 31, 50 and 76K in the flagellar roots ofDunaliella. In 1984, Salisbury and co-workers obtainedan antiserum raised against a 20K calcium-binding pro-tein, centrin, which is present in Tetraselnns. Thisprotein was also found to be present in the flagellar rootsof Chlamydomonas and Polytomella (Salisbury et al.1986; Huang et al. 1988). Recently, Baron and Salisbury(1988), using the same serum anti-centrin, observed a165K protein immunologically related to centrin in PtK2cells. This protein is present in pericentriolar materialand the basal foot, but not in striated rootlets. In scallopgill, Stephens (1975) found two major proteins of 230 and250K in the striated rootlets. Monoclonal antibodyCC310 recognized, after immunofluorescence, striatedrootlets of different epithelial cells, i.e. from mussel gill,frog palate, human broncheal biopsies and rootlets fromPtK2 cells (Klotz et al. 1986). With the same antibody,


••«•'*.

4C

Fig. 4. A-C. Immunogold staining of ciliated cell with polyclonal antibody. X67 500. After pre-embedding immunodetection(A and B) gold granules are distributed along the striated rootlets (sr). When rootlets are only slightly extracted by Triton X-100 (A), the apical part is poorly decorated (arrow). In Triton X-100-extracted rootlets (B) the striations have disappeared andthe fibrillar axis (arrows) is uniformly decorated by gold granules. No other ciliary or cellular structure is decorated. After apost-embedding immunocytochemical study (C), gold granules are observed on the striated rootlets (sr), irrespective of thestriation, and irrespective of the apical or basal part of the rootlet. Basal body (bb) and axoneme (a) are unlabeled.Fig. 5. Pre-embedding immunogold labeling of ciliated cells using monoclonal antibody CC310. X67 500. We can observe arandom deposit of gold granules along the striated rootlet (sr). Dense granules associated with cytokeratin filaments are alsostrongly decorated (arrow). Basal bodies (bb), basal foot (bf) and anchoring fiber (af) are weakly labeled.

CC310, we demonstrated the conservation of an epitopeassociated with striated rootlets during evolution, but themolecular weight and distribution of detected antigens on

striated rootlets changed with the different species stud-ied (Lemullois et al. unpublished data).

We cannot establish a relationship between the 175K


Fig. 6. Immunogold staining of the diplosome in an epithelial cell before terminal differentiation. X67 500. The two centrioleslie in tandem orientation. The distal centriole (dc) is associated with the apical membrane. Gold granules are localized aroundthe proximal centriole (pc) and the thin striated rootlets (sr).Fig. 7. Immunogold staining of a ciliogenic cell at the beginning of centriologenesis. X67 500. The two pathways ofcentriologenesis are observed. In the centriolar pathway, the centrioles of the diplosome (d) are surrounded by dense materialand dense granules (g). At the bottom of the micrograph, a deuterosome (D) begins to form by the merging of dense granules(g). Only the striated rootlet (sr) of the diplosome and fibrillar material (arrow) distributed between the dense granules aredecorated by gold granules.

protein detected by our polyclonal antibody and thedifferent proteins described above, since this polyclonalantibody recognized one or several epitopes only, in theciliated cells of quail. In an immunofluorescence study,we obtained no results with mussel gill, rat tracheal orhuman biopsies.

Concerning the building of striated rootlets duringciliogenesis, the 175K protein does not seem to bepackaged in the dense granules observed near the Golgi

apparatus and the deuterosomes. The 175K proteinappears prior to centriologenesis between these densegranules. During centriologenesis, the 175K protein isobserved surrounding the generative complexes (deuter-osome and nascent procentrioles). Once the kinetosomeis formed and leaves the deuterosome, an early section ofthe rootlet set up at the basal pole of the kinetosome. Atthis stage, the kinetosome has already acquired itspolarity. The elongation of rootlets takes place exclus-


m

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D

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rV

. bf

, af

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Fig. 8. Immunogold staining of generative complexes during the acentriolar pathway of centriologenesis. X67 500. Goldgranules are distributed around the procentriole (pc) without clear orientation. The deuterosome (D) and procentriole cores areunlabeled.Fig. 9. Immunogold staining of ciliogenic cortices during migration and anchoring phase of basal bodies. X67 500. Goldgranules are localized on fibrillar material (arrows) associated only with the basal pole of migrated and anchored basal bodies(bb).Fig. 10. Immunogold staining of elongating rootlets. X67500. The nascent rootlets (sr) are labeled. Dense granules (g)localized under the basal bodies (bb) are not decorated by gold granules. Axoneme (a), basal foot (bf) and anchoring fibers (af)are also unlabeled.


ively when the kinetosome is anchored to the membrane.It seems that the two groups of components of striatedrootlets, i.e. the fibrillar axis and the striation material,merge. In no case did we observe formation of thefibrillar axis followed by deposit of the striated material.

In quail oviduct there exists no intracellular centerspecialized for the formation of striated rootlets, as wasdescribed in chicken trachea by Kalnins and Porter(1969). In quail oviduct, the elongation of striatedrootlets seems to be associated with the anchorage ofkinetosomes to the membrane as already described(Chailley et al. 1982). In contrast, in the CtenophoreBeroe macrocilia cells, the formation of striated rootletsoccurs once the centrioles are formed and prior to theirmigration (Tamm and Tamm, 1988). In 1971, Andersonand Brenner suggested that components of striated root-lets come from dense granules under the basal bodies. Wecannot confirm this hypothesis, since dense granules arenot labeled by our antibody. They could contain thecomponent of dense material that produces striation.

In macrocilia cells of Beroe (Tamm and Tamm, 1988),the basal body-rootlet units are in close association withactin bundles during the migration phase. In quailoviduct, this same phase seems to be actomyosin-depen-dent, since actin (Lemullois et al. 1988) and myosin(Lemullois et al. 19876) appear to be associated with themigrating kinetosome; however, we did not observe aclose association between the forming rootlets and actin.

We conclude that the 175K protein is one of thecomponents of striated rootlets. This protein forms themain filamentous axis of these rootlets. It appears earlyduring centriologenesis, but the elongation of striatedrootlets occurs only after basal body binding to the apicalmembrane.

I wish to thank Dr D. Sandoz for his stimulating discussionand his continuous interest throughout this work; without hisgenerous support this study would not have been possible. Weare grateful to Dr C. Klotz (Centre de Ge'ne'tique Mole'culaire,Gif sur Yvette, France) for the gift of the monoclonal antibodyCC310. This work was supported by a grant from the Fon-dation pour la Recherche Me'dicale. We thank M. Louette forphotographic work and P. Gaudoin for typing the manuscript.

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immunocytochemical study of the formation of striated ...(1975). these solutions were sonicated at...

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