Cell Motility and the Cytoskeleton 17:317-328 (1990)

Identification of Surface Components of Mammalian Respiratory Tract Cilia Annette T. Hastie, Mori J. Krantz, and Frank P. Colizzo

Department of Medicine, Thomas Jefferson University Philadelphia, Pennsylvania Cilia isolation methods were modified to retain respiratory tract ciliary membranes and to identify accessible surface components. Prior to isolation of cilia, halves of cow tracheae were treated with the extended spacer arm analog of N-hydroxysuccinimido-biotin (NHS-LC-biotin) to label accessible membrane constituents. Mechanical disruption of the epithelium and substitution of CHAPS for Triton X-100 provided a good yield of cilia with membranes and with minimal contamination. Subsequent extraction of these cilia with Triton X- 100 solubilized the membranes and released soluble matrix proteins. Proteins of membrane + matrix and axoneme fractions were analyzed after electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The major biotin-labeled components in the membrane+matrix fraction were 105, 98, and 92 kd, were glycosylated, and remained with reconstituted, pelleted membrane vesicles along with the major non-biotinylated protein at 5 1 kd. Other membrane matrix proteins at 126 and 76 kd bound streptavidin even from noniabeled trachea, but remained soluble. Several biotin-labeled proteins distinct from those in the membrane fraction remained with Triton X- 100-extracted axonemes. Streptavidin-colloidal-gold (SAG) particles appeared to bind randomly along the length of cilia. The peripheral join between A and B microtubules was a predominant nonspecific location of SAG on axonemes. Axonemes with biotin label also bound significant numbers of SAG to outer dynein arms, confirming the streptavidin reaction with separated proteins on transfers. These results suggest close association of the membrane with the axoneme in respiratory tract cilia and a membrane composition somewhat different from protozoan cilia.

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Key words: bovine trachea, cilia, axonemes, ciliary membranes, biotin-streptavidin, colloidal-gold

INTRODUCTION Membrane components of respiratory ciliated cells which are accessible to the environment may interact with secretory products, inhaled material, or microbes and their exoproducts. Such interaction evokes a response in ciliary function either stimulatory [Spungin and Silberberg, 1984; Sanderson and Dirksen, 19861 or inhibitory [Tuomanen and Hendley, 1983; Hingley et al., 19861. Although membranes of protozoan cilia [Adoutte et al., 1980; Dentler et al., 1980; Dentler, 1988a,b] and flagella [Bloodgood, 19871 have been analyzed, mammalian ciliary membranes have received little attention [Chen and Lancet, 19841. Adequate numbers of ciliary axonemes can be obtained from mammalian respiratory 0 1990 Wiley-Liss, Inc.

epithelium through the use of Triton X-100 and CaCI, but with consequent loss in the extraction process of the membranes [Hastie et al., 19861 which may be mixed with secreted and intracellular proteins released upon membrane disruption. Therefore, an alternate method was sought for isolation of respiratory tract cilia by which their surrounding membranes would be retained

Received January 16. 1990; accepted August 3 , 1990. Address reprint requests t o Dr. Annette T. Hastie, Department of Medicine. Thomas Jefferson University, 1025 Walnut Street, Room 804 College Building, Philadelphia, PA 19107.

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and surface components identified. These cilia could be subsequently separated into membrane and axoneme fractions, the membrane fraction containing in addition the internal matrix proteins released by lipid bilayer solubilization [Gibbons, 19651. To identify components in the membrane matrix fraction which were originally accessible at the epithelial surface, biotin labeling was employed prior to isolation of cilia [Reinhart and Bloodgood, 19881. Membrane vesicles were reconstituted and sedimented from the membrane matrix fraction to distinguish proteins predominantly associated with the vesicles from those proteins remaining in solution.

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MATERIALS AND METHODS Isolation of Cilia and Triton X-I00 Extraction of Membranes

Bovine tracheae were obtained and transported to the laboratory within 1/2 hour from time of sacrifice at local slaughterhouses under permit by the U.S. Department of Agriculture. The epithelium of saline-rinsed, one-half bovine trachea was lightly brushed with a clean nylon bristle brush to disrupt the epithelium. The brush bristles were rinsed with 75 ml of extraction buffer containing CHAPS (Sigma Chemical Company, St. Louis, MO) (0.1% CHAPS, 50 mM NaCl, 20 mM Tris-HC1, pH 7.5, 1 mM EDTA, 7 mM 2-mercaptoethanol, and 10 mM CaCI,) which was subsequently poured into the tracheal lumen and shaken vigorously for 30 sec. The suspension of cellular debri and cilia was retained and added to 75 ml of extraction buffer without CHAPS which was used to rinse the trachea. The 150 ml suspension was centrifuged at 2,OOOg for 5 min. The supernate was decanted and centrifuged at 12,OOOg for 5 min. An aliquot of the extraction buffer was saved for sodium-dodecylsulfate polyacrylamide gel electrophoretic analysis (SDS-PAGE). The pellet containing isolated cilia and some cellular debri was resuspended in 1 ml of resuspension buffer (RB; 50 mM potassium acetate, 20 mM Tris-HCI, pH 8.0, 4 mM MgSO,, 1 mM dithiothreitol, 0.5 mM EDTA) and repelleted at 12,OOOg for 1.5 minutes. The pellet was resuspended in 4 ml of RB with 0.1 mgiml soybean trypsin inhibitor, and recycled through the centrifugation steps, first at 2,0001: for 1.5 min and then 12,OOOg for 1.5 min, to remove contaminating cellular debri. The final pellet was resuspended in l ml of RB with soybean trypsin inhibitor. A 0.6 ml aliquot was spun at 12,00013 to pellet cilia which were then resuspended in 0.25 ml of 0.5% Triton X-100 in RB with soybean trypsin inhibitor for 15 min to solubilize ciliary membranes. Demembranated ciliary axonemes were pelleted at 12,00Og, rinsed, and resuspended in 0.3 ml of RB with soybean trypsin inhibitor.

The supernate containing the Triton X- 100-soluble material was retained as the membrane matrix fraction.

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Reconstitution of Membrane Vesicles

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Triton X- 100 membrane matrix supernates were processed to remove the detergent by use of Extracti-Gel D (Pierce, Rockford, IL). A volume of 0.75 ml of nonlabeled or biotin-labeled membrane matrix solution was placed on a 2 ml column of Extracti-Gel D, eluted with RB, and collected in 0.5 ml fractions. The two fractions containing peak protein concentration were combined, frozen, and thawed. The reconstituted membrane vesicles were pelleted at 48,700g for 15 min. The supernate was retained for SDS-PAGE analysis, and the pellet resuspended in 0.2 ml of RB with soybean trypsin inhibitor.

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Biotin Labeling of Tracheal Epithelial Surface Components

Bovine tracheae were divided into equal halves. The control half was incubated in 60 ml of phosphatebuffered saline (PBS; 0.12 M NaCl, 2.7 mM KCI, 5 mM potassium phosphate, pH 7.4) for 30 min on a rotary platform (100 RPM) at room temperature. The other half was incubatcd in 15 mg NHS-LC-biotin (Pierce, Rockford, IL) in 60 ml of PBS under the same conditions. Each tracheal half was rinsed briefly with two 50 ml aliquots of sterile saline before isolation of cilia. To assess the effect of the biotin labeling procedure on ciliary function of intact epithelium, two rabbit tracheal rings were incubated in 0.1 ml of PBS with 0.25 mg NHS-LC-biotidml and one tracheal ring in 0.1 ml of PBS alone as a control for 30 min. Active ciliated epithelium viewed by light microscopy in the same two separate zones was videorecorded before and at 10 min intervals after the addition of the biotin solution. These zones were examined for sites of decreased or inhibited ciliary activity as described in detail [Hastie et al., 19871. The experiments were repeated with three different rabbits. In more recent experiments, a small piece of the epithelium from the bovine trachea in use was concurrently incubated in the biotin solution and examined by light microscopy for continued activity throughout the labeling period. For preparation of ciliary axonemes labeled with biotin after removal of their membranes, aliquots of 0.25 ml of Triton X- 100-extracted ciliary axonemes were pelleted at 12,OOOg, resuspended in 1 ml of PBS with 0.25 mg NHS-LC-biotin, and incubated for 30 min at room temperature. The axonemes were pelleted, washed, and resuspended in 0.25 ml of RB with soybean trypsin inhibitor.

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were added directly to the suspension of SAG. The suspensions were gently agitated for 1 hr at room temperaSamples to be examined by SDS-PAGE were diture. After incubation with streptavidin-gold, the cilia, luted 1: 1 with sample buffer and heated at 100°C for 2-3 extracted axonemes, and membrane vesicles were pelmin before loading on a 4-10% polyacrylamide gradient 3 min, and washed in HCMNT leted at 12,OOOg for separating gel with 3% stacking gel. Standard SDS displ volume of HCMNT buffer containing buffer. A 200 sociating, high pH discontinuous buffers [Maizel, 197I ] 2.5% glutaraldehyde was added to each of the final pelwere employed with the exception of the electrode buffer lets. Pellets were postfixed in osmium tetroxide, dehywhich was 25 mM Tris, 0.2 M glycine, and 0.1% SDS. drated, and embedded in Spurr’s low viscosity medium. After an initial migration through the stacking gel at 20 Thin sections were stained with uranyl acetate and lead mA, electrophoresis was at 60 mA with cooling at 16°C. 80 kV in a Zeiss EM 109 eleccitrate, and examined at Gels were stained with Coomassie brilliant blue or periX magnification or at 75 kV tron microscope at 28,800 odic acid-Schiff (PAS) [Fairbanks et al., 19711 or were transferred to Immobilon (Millepore Corp., Bedford, in a Hitachi H7000 electron microscope at 8000MA). Gels were rinsed for 20 min and then transferred at 30,000 X magnification. Quantitation of SAG particles on Triton X-100 ex100 V to Immobilon in 25 mM Tris, 0.2 M glycine for 1 tracted axonemes was obtained from 16 micrographs per hr. The Immobilon transfers were blocked with nonfat sample in two separate experiments. Only axonemes obmilk [Johnson et al., 19841 and rinsed with H,O. To served in direct cross-section were scored where the podetect biotin-labeled components the transfers were insition of SAG adherence could be unequivocally attribcubated for 1 hr in a 1 5 0 0 dilution of streptavidin-biotinuted. The A-B position was defined as located at the horseradish-peroxidase complex (Amersham Corp., Arperipheral join between A and B subfibers of an individlington Heights, IL) in HCMNT buffer (10 mM HEPES, ual doublet microtubule; the dynein position was located pH 8.0, 0.1 mM CaCl,, 0.1 mM MnCI,, 0.15 M NaCl, at the outer dynein arm; and other was any other location 0.1% Tween 20) with 1% gelatin. The transfer was such as central microtubules. Chi-square statistical analrinsed with HCMNT buffer, 3 X 10 min, once rapidly ysis of results was performed. with H,O, and developed with 4-chloro- 1-napthol. Alternatively, to determine the correlation between strepta- Protein Concentration and ATPase vidin-biotin-HRP complexes and streptavidin-colloidal- Activity Determination gold reagents, the transfer was incubated in streptavidinAliquots of isolated cilia, Triton X- 100-extracted colloidal-gold, 10 nm size (E-Y Laboratories, San axonemes, and Triton X- 100 membrane matrix superMateo, CA) for 1 hr in the same buffer conditions. After nates were assessed for protein concentration and rinsing, the transfer was developed in the dark by a modATPase activity. Protein concentration was determined ified Danscher method for silver enhancement of gold by the Bradford method [1976] using BioRad Protein label [ 198I]. Stained gels and transfers were scanned on Assay Reagent micromethod (BioRad, Richmond, CA) a Hoefer GS 300 Scanning Densitometer (Hoefer Scienwith ovalbumin as a standard protein. ATPase activity tific Instruments, San Francisco, CA). was assessed by the measurement of inorganic phosphate [Fiske and Subbarrow, 19251 after incubation of exLight and Electron Microscopy tracted axonemes, and Triton X- 100 soluble membrane Aliquots of the isolated cilia, after the first and matrix fraction with ATP as described in Hastie et al. second cycles of differential centrifugation steps, and of [1986]. Triton X- 100-extracted ciliary axonemes were examined for contamination and reactivation upon ATP addition by RESULTS phase contrast light microscopy coupled with videoreSeveral alternatives to the Triton X- 100 procedure cording at a final magnification of 4,050 x as described in Hastie et al. [1986]. Aliquots of reconstituted mem- were explored in order to retain the ciliary membrane. brane vesicles were also examined for contamination by The modifications that provided the best yield of cilia were the use of an extraction buffer with 0.1% CHAPS, phase contrast light microscopy. Fifty pl aliquots of isolated cilia, and Triton X- 100 a zwitterionic detergent, coupled with brushing of the extracted axonemes were pelleted at 12,000g and resus- epithelium. The use of extraction buffer with CHAPS pended in 200 p1 HCMNT buffer containing 0.1 mM without mechanical disruption was less productive, phenylmethylsulfonyl fluoride, 2 pg/ml leupeptin, 0.1 yielding only one-tenth the protein and ATPase activity pg/ml pepstatin, a I / 10 dilution of streptavidin-colloidal- in the axoneme fractions and one-fifth the protein in the gold (SAG), 10 nm, and either 1% gelatin or 0.25 M membrane matrix fractions. The ATPase activity in the sucrose. Aliquots of reconstituted membrane vesicles membrane matrix fraction from nonbrushed tracheal SDS-PAGE

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Fig. 1. Light micrograph (a) and electron micrograph (b) of isolated cilia preparation. Cellular debri, in the form of large phase-dark particles in the light micrograph, appeared as loose aggregates of reticular material and membrane vesicles in electron microscopic thin section. The isolated cilia were, however, the predominant structure present. The bar represents 10 pm in the light micrograph and I pm in the electron micrograph.

responding halves treated with the Triton X- 100 method [Hastie et al., 19861. Cilia isolated by the CHAPS extraction buffer and brushing procedure retained surrounding bilayer membranes (Fig. 2) which were solubilized by subsequent treatment with Triton X-100 (as seen in Fig. 6). The dense matrix filling the internal axoneme structure was further evidence of substantially intact membranes on these cilia. This dense matrix was absent in Triton X100-extracted axonemes (Fig. 6) and thus contained in the membrane + matrix fraction. In addition, the majority of these cilia did not reactivate to beat in suspension with ATP, until after extraction with Triton X-100. Some of the material contaminating the isolated cilia preparation may also be solubilized by the Triton X-100 extraction. In order to distinguish between surface membrane components from the cilia, contaminants, and other materials such as the internal matrix proteins released by membrane solubilization, the tracheal epithelial surface was labeled with biotin prior to isolation of cilia and extraction of membranes. Proteins released from the trachea into the solution during incubation with biotin were also labeled. Biotin labeling did not inhibit ciliary activity of representative segments from bovine tracheal epithelium or rabbit tracheal epithelium during a 30 min incubation period. The Triton X- 100-soluble membrane + matrix fraction and extracted ciliary axonemes from biotin-labeled and nonlabeled tracheal epithelium were assessed for protein concentration and ATPase activity (Table I). There were no significant differences in comparing the biotin-labeled with the nonlabeled axonemes and membrane + matrix fractions. The membrane + matrix fraction contained 12.5-14% of the total Mg*+-ATPase activity from axoneme and membrane matrix fractions. The specific ATPase activity for the Triton X-100-extracted bovine axonemes is at the lower end of the range found for porcine axonemes [Hastie et al., 19861. The Triton X- 100-soluble membrane matrix fraction and extracted ciliary axonemes from 5 different tracheae were examined on SDS-PAGE (Fig. 3). The migration of proteins stained with Coomassie brilliant blue (CBB) in either membrane + matrix or axoneme did not differ between biotin labeled or nonlabeled samples. There were few, very faint protein bands above 110 kd in the membrane + matrix fractions, and no one protein was predominant at lower molecular weights. The two bands of heaviest density at 51 and 47 kd comprised only 4.5 and 6.5% of the total, respectively. Although bands in the membrane + matrix samples migrated equivalent to the heavily stained tubulin subunits in the extracted axoneme samples, none reacted with anti-tubulin antibody. The tubulin subunits and high molecular weight dynein bands constituted 40.7 and 7.4%, respectively, of the

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epithelium was negligible. Although brushing the epithelium versus not brushing increased the yield of isolated cilia, it may also contribute greater cellular debri (Fig. 1) and perhaps losses through the consequent necessity of repeating the differential centrifugation steps to reduce contamination in the isolated cilia fraction. However, the specific ATPase activities in the axoneme fractions derived from CHAPS extraction buffer and brushing technique compared to those derived without brushing were the same. Other agents such as Nonidet P40, DMSO, or sucroseiethanol produced insufficient yields of cilia. Halves of four tracheae, two biotin-labeled and two nonlabeled, each treated with the CHAPS extraction buffer and brushing procedure yielded equivalent amounts of ciliary axoneme protein and ATPase activity as the cor-

Mammalian Ciliary Surface Components

Fig. 2. Electron micrographs of isolated cilia, biotin-labeled (a) and nonlabeled (b). The isolated cilia were surrounded by a bilayer membrdne which occasionally was stripped away, probably by mechanical means. Also present in the preparation of isolated cilia was some cellular debri. Both biotin-labeled (c) and nonlabeled preparations (d) were incubated with SAG particles (arrowheads). which adhered. in

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seemingly random fashion, more frequently to biotin-labeled cilia. The nonlabeled axoneme without a membrane in d bound SAG (see Fig. 7). Micrographs in the a and b pair, and in the c and d pair were printed to the same magnification; the bar for each pair represents 100 nm.

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TABLE I. Extracted Axoneme and Membrane+ Matrix Protein and ATPase Values*

Total Drotein

Samole Axoneme Biotin Biotin-

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Specific ATPase activitv

0.45 i .08" 216 0.40 t .07 231

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Membrane matrix Biotin + 0.44 2 .04 Biotin0.45 0

*

2

c/c of combined Total ATPase ATPase (Ax & Mb)

2 I b 98 t 26' 94 2 28

* 39

32 t 18 34 6

*

14 t 9 15 2 3

87.5 86

12.5 14

*The mean ? standard deviation from three experiments. There were no significant differences between labeled (biotin + ) and nonlabeled (biotin-) axonemes or membranes. "Values given in mg. 'Values given in nmoles P, releasedlrninimg protein. 'Values given in nmoles P, releasedimin.

total in the extracted axoneme fractions. Some bands migrated identically in both membrane matrix and axoneme fractions. These may represent axoneme proteins partially solubilized by Triton X- 100, membrane matrix proteins incompletely solubilized, or different proteins in the two fractions which have similar molecular weight. Transfers of the membrane matrix fraction tested for biotin label by streptavidin (SA) revealed that the major biotin-labeled components were at 105,98, and 92 kdaltons which did not stain heavily with CBB (Fig. 3). Two bands, one at 126 and a doublet at 76 kd, in the membrane matrix fraction bound SA even without prior biotin labeling. In fractions which were biotin-labeled, the amount of SA binding to these proteins appeared increased, suggesting that the 126 and 76 kd proteins may also become biotin-labeled. Membrane matrix fraction proteins at 5 1 and 47 kdaltons had little, if any, biotin label. Many proteins remaining with the Triton X- 1OO-extracted axonemes were biotin-labeled and distinct from those in the membrane+matrix fraction. Those consistently having biotin label in all preparations included proteins at >300, 200, 180, 150, 72, 53, 50,42, and 32 kdaltons. Axonemal tubulin from cilia labeled with biotin prior to isolation was negligibly labeled with biotin, but the high molecular weight dynein proteins (>300) were labeled with variable amounts of biotin. For comparison, ciliary axonemes biotin-labeled after Triton X- 100 extraction were also examined on SDS-PAGE (Fig. 4). Although proteins in the CBBstained ciliary axoneme samples were nearly identical with those which had been labeled prior to isolation (see Fig. 3, 4), the biotin-labeled components were different.

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These results suggest that the majority of proteins in the axoneme after Triton X- 100 extraction were accessible for biotin labeling, but that in the intact cilium with surrounding membrane only certain components were accessible. The membrane matrix fractions were processed to reconstitute membrane vesicles. These were sedimented in order to determine which proteins remained with the membrane vesicles and which remained in solution. The sedimented material consisted of membrane vesicles of varying sizes (Fig. 5). The total ATPase activity of four membrane + matrix fractions divided approximately equally into the pellet of reconstituted membranes and into the supernate, though the specific ATPase activity was higher in the membrane pellet. SDS-PAGE analysis of the original membrane matrix fraction, the pelleted membrane vesicles, and the supernate showed segregation of proteins comprising the membrane + matrix fraction (Fig. 6, CBB). Many proteins remained in the supernate and did not appear in the membrane pellet despite a 5-fold concentration in resuspension of the membrane vesicles. The presence of other proteins, in particular those at 160, 107, 97, 69, 51, 47, 43, 38, 33, 28, and 23 kd, appear in substantially greater amounts in the membrane pellet. By concentrating the membrane vesicles, these proteins were correspondingly concentrated and, therefore, were presumably membrane constituents. The 51 kd protein was now the predominant protein in the membrane pellet. A transfer for detection of biotin-labeled materials by streptavidin (Fig. 6, SA) showed segregation of labeled components at 105, 98, 92, and 80 kd into the membrane vesicle pellet, whereas the two proteins, at 126 and 76 kd, described above as binding SA even without biotin labeling, remained primarily in the supernate. The diffuse spread of the components ranging from 105 to 80 kd was attributed to carbohydrate content, and confirmed by PAS staining (Fig. 6). The biotin-labeled membrane matrix and membrane vesicles shown here were obtained from a different trachea than the nonlabeled samples, which may account for the difference in amount of PAS-stained material migrating slightly further in the nonlabeled samples. Isolated cilia, reconstituted membrane vesicles, and Triton X- 100-extracted axonemes which were biotinlabeled or nonlabeled were treated in suspension with streptavidin-colloidal-gold (SAG) particles to locate the sites of biotin label (Fig. 2, 5, 7). SAG particles adhered to non-biotin-labeled cilia, membrane vesicles, and extracted axonemes despite suspension in 1% gelatin which successfully blocked non-specific binding of streptavidin to the separated proteins on transfers with the exception of the membrane + matrix components at 126 and 76 kd. However, fewer numbers of SAG adhered to nonlabeled

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Mammalian Ciliary Surface Components SA Membranes a

b c d c a’ b’ c’ d’ c’

I

o

CBB Membranes

CBB Axonemes

b c

b e d c a’b’c’d’e’

d c

a’b’c‘d’c’o

323

SA Axonemes a b c d c a‘ b’ c‘ d’ c ’

I

200

200

116

116

93

93

68

68

45

45

Fig. 3. SDS-PAGE of ciliary membranefmatrix and Triton X-100extracted axoneme fractions from five tracheae (a+), half of which were biotinylated (a‘+’). Samples were stained for protein with Coomassie brilliant blue (CBB). Molecular weight standards (s) were myosin (200 kd), beta-galactosidase (1 16 kd), phosphorylase b (93 kd), bovine serum albumin (68 kd), and ovalbumin (45 kd). Duplicate samples of membrane and axoneme fractions were transferred and developed for biotin detection with HRP-streptavidin (SA) in 1% gel-

atin. Prestained molecular weight standards on right and left edges of transfers were 217, 103, 69, 49, and 32 kd. The major biotin-labeled components of the membrane +matrix fractions were at 105, 98, and 92 kd which did not stain heavily with CBB. Two components in the nonbiotinylated membrane fractions at 126 and 76 kd reacted nonspecifically with SA. Many biotin-labeled proteins identified by SA, including the dynein proteins at >300 kd, remained in the Triton X100-extracted axoneme fractions.

compared to biotin-labeled cilia, membrane vesicles, and extracted axonemes. There did not appear to be a preferred location along the length or at either end of isolated cilia for binding, suggesting that surface components carrying biotin label were dispersed rather than localized in a particular region. The peripheral join between the A and B subfibers of the outer doublet microtubules was the predominent location of the SAG particles adhering to both biotinlabeled and nonlabeled Triton X- 100-extracted axonemes (Fig. 7). Quantitation of the SAG particle binding sites is given in Table 11. There were greater numbers of biotinlabeled axonemes with adherent SAG particles than of nonlabeled axonemes, significant in one experiment ( P < .05) and of borderline significance in the other (P = .OSS). Also there were significantly greater numbers of SAG particles adhering to the outer dynein arm position in the biotin-labeled axonemes than in nonlabeled axonernes ( P = .02 and P < .001 in the two experiments) corresponding to biotin labeling of dynein proteins as observed on SDS-PAGE transfers (see Fig.

3 ) . The join between the A and B subfibers was, nevertheless, the most frequent position. DISCUSSION

Methods previously described for cilia isolation which did not employ a detergent, including the use of dibucaine [Thompson et al., 19741, or sucrose/ethanol/ CaCI, [Gibbons, 19651, were tried in order to retain the ciliary membrane. However, replacement of Triton X100 with CHAPS detergent at a concentration below its critical micelle concentration [Hjelmeland et al., 19831 and mechanical trauma in the form of brushing the epithelium proved effective for isolation of cilia retaining a surrounding membrane. Sufficient amounts were obtained by this procedure for subsequent fractionation into membrane matrix and axonerne components. The brushing also introduced greater contamination which could be reduced but not eliminated by repetition of the differential centrifugation steps. Further purification steps would probably decrease contaminants in the iso-

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SA g g’ Sl

h h‘hl

CBB g g‘gl h

h‘ hl s

217

103

69

49

32

Fig. 4. SDS-PAGE of ciliary axonemes from two tracheal preparations (g,h) biotin labeled (g’-h’) after Triton X-100 extraction of membrane matrix. Biotin label incubaton buffer ( I ) was also examined in each. Molecular weight markers ( s ) were myosin (217 kd), phosphorylase b (103 kd), bovine serum albumin (69 kd), ovalbumin (49 kd), and alpha-chymotrypsinogen (32 kd). There was no noticeable difference in protein migration, stained with Coomassie brilliant blue (CBB), after biotin label addition compared with nonlabeled proteins. All proteins appeared to be biotin-labeled, identified with streptavidin (SA) though some label was greater in proportion than protein concentration.

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lated cilia suspension, but handling through numerous resuspension steps removes the membranes from isolated cilia [Adoutte et al., 19801 and thus additional steps were avoided. The cilia were, nevertheless, the predominant structure present. Biotin labeling was used to identify components in the isolated cilia and their membrane and axoneme subfractions which had been accessible at the epithelial surface. Biotin label did not interfer with ciliary function of intact cells or isolated axonemes. Proteins released from the epithelium into the label incubation buffer became biotinylated. Some of the released proteins may adher to

ciliary surfaces and remain through the isolation procedure, although this is considered unlikely given the thorough rinsing performed after labeling, and vigorous agitation during extraction of cilia from the epithelium. In addition, the use of detergent in the extraction buffer would probably lessen nonspecific association of secreted proteins with the epithelial surface. Negligible biotin-labeled material was found in the extraction buffer employed for bovine cilia isolation, suggesting that the CHAPS detergent was solubilizing very little of the surface components. This result may be compared to Stephens’ observation that Nonidet P40, at a concentration up to 4 times its critical micelle concentration [ 19851, solubilized very little membrane protein from scallop gill cilia, leaving a membrane sleeve on the axonemes [Stephens et al., 19871. The major biotin-labeled membrane components were 105, 98, and 92 kd, though they did not comprise a major portion of the total membrane protein. The diffuse migration of these components suggests that they may be glycosylated, and indeed, corresponded to PASstained material and with material which strongly binds Dolichos biflorus lectin [Hastie and Krantz, 19881. A protein of 100 kd is one distinctive constituent of the ciliary membrane of Aequipecten [Stephens, 1977; Dentler et al., 19801 and a protein from Tetrahymena mcmbrancs of 104 kd is found in Triton X-114 aqueous and, more prominently, detergent phases [Dentler, 1988al. However, the 104 kd Tetrahymena protein [Dentler, 1988al and the 100 kd Aequipecten protein [Stephens, 19771 do not appear to be heavily glycosylated as is the 105 kd component of bovine ciliary membranes. The major protein of reconstituted bovine ciliary membranes was 51 kd, but was not biotin-labeled, did not stain with PAS, and did not react with anti-tubulin antibody. A 50 kd protein in the membrane+matrix fraction weakly bound certain lectins suggesting minor carbohydrate content [Hastie and Krantz, 19881. These results indicate that this protein, although a component of ciliary membranes, is inaccessible or unreactive with the biotin label, contains little carbohydrate, and is not tubulin. These features correspond with certain aspects but are dissimilar to others of ciliary membranes in various species. Tubulin was not prominent in ciliary membranes from frog respiratory epithelium [Chen and Lancent, 19841 and Paramecium tetraurelia [Adoutte et al., 19801, but it was the major component in Aequipecten [Stephens, 1977; Stephens, 19851. Tubulin was found in the Triton X-114 aqueous phase but not the detergent phase of Terrahymena ciliary membranes [Dentler, 1988al. A 50 kd protein was one of two major components of the Tetrahymena ciliary membranes. However, the 50 kd protein is glycosylated and is labeled with a

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Fig. 5. Electron micrographs of membrane vesicles reconstituted from biotin-labeled (a) or nonlabeled (b) membrane + matrix fractions. Membrane vesicles of varying sizes were observed in sedimented material. Suspensions of biotin-labeled ( c ) and nonlabeled (d) membrane vesicles were incubated with streptavidin-gold 10 nm par-

ticles. Gold particles (arrowheads) were observed more frequently in the biotin-labeled preparation of membrane vesicles than in the nonlabeled vesicles. All micrographs were printed to the same magnification; the bar represents 100 nm.

biotinylated probe [Dentler, 1988a,b] or similarly, a 48 [Williams et al., 19801 in kd protein is labeled with living Tetruhymena cells, indicating surface accessibility. Therefore, the 51 kd protein of bovine ciliary membranes is different from equivalently sized components of protozoan ciliary membranes. There was some minor ATPase activity in the bovine respiratory membrane matrix fraction which divided approximately equally into the membrane vesicle

pellet and supernate subfractions. ATPase activity of Tetruhymenu ciliary membranes was primarily associated with the detergent phase [Dentler, 1988al. There was no bovine ciliary membrane protein in the high molecular weight size of outer arm dynein as occurs in Aequipecten [Stephens, 1977; Dentler et al., 19801 and Tetruhymenu [Dentler, 19801, although the Tetruhvmenu high molecular weight protein remained in the Triton X-114 aqueous phase, separate from the main membrane

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PAS

SA

t'p't p

ht's'p'h t s p

CBB t's'p't s p m

4217

4103 4

69

4

49

4

32

;2 4 Fig. 6. SDS-PAGE of original membrane + matrix fractions (t), and subfractions of supernate (s) and sedimented membrane vesicles (p) obtained from membrane reconstitution of biotin-labeled (') and nonlabeled material (no '). Molecular weight markers (m)were myosin (217 kd), phosphorylase b (103 kd), bovine serum albumin (69 kd), ovalbumin (49 kd), alpha-chymotrypsinogen (32 kd), and beta-lactoglobulin (24 kd). Replicates of the samples were stained with Coomassie brilliant blue (CBB), periodic acid-Schiff (PAS) for detection of carbohydrate, or streptavidin (SA) for detection of biotin label. One-half the amount of the biotin-labeled and nonlabeled membrane matrix samples was loaded (h' and h) to reduce overlap but did not increase resolution of biotin-labeled components detected by streptavidin (SA). Certain proteins including those of 126 and 76 kd which nonspecifically bound SA, remain primarily in the supernate, whereas others, including the biotin-labeled components at 105, 98, and 92 kd in addition to several nonlabeled proteins, segregate predominantly into the pelleted membrane vesicles.

+

ATPase activity [Dentler, 1988al. However, ATPase activity associated with other membrane proteins is a possibility [Dentler, 1988al. Despite Triton X-100 solubilization of the ciliary membrane bilayer, several biotin-labeled proteins remained with the axoneme, indicating resistance to detergent extraction. Reinhart and Bloodgood [ 19881 observed a similar association of biotin-labeled flagellar surface components with the axoneme fraction after detergent extraction. Similarly, a cell surface glycoconjugate stayed attached to photoreceptor cilia in rat retina

Fig. 7. Electron micrographs of Triton-extracted axonemes from trachea, biotinylated (a) and nonbiotinylated (b), prior to isolation of cilia. Ciliary axonemes were incubated with streptavidin-gold 10 nm particles before embedding and sectioning. The gold particles adhered to the axonemes, both biotin-labeled and nonlabeled. The predominant location of the gold particle in the cross-sectioned axoneme was at the peripheral join of A and B doublet microtubules. Gold particles also adhered to outer dynein arms in biotin-labeled axonemes more frequently than in nonlabeled axonemes. Micrographs are printed to the same magnification; bar represents 100 nm.

after phospholipid bilayer removal [Horst et al., 19871. From these observations, it seems reasonable to conclude that certain surface components of cilia are detergent insoluble, and preferentially associate with the internal axoneme. In the present experiments, however, proteins corresponding to the dynein arm, an internal structure, became labeled with biotin during incubation of intact epithelium. The variable labeling of the dynein proteins obtained here suggests that ciliated cell membranes in

Mammalian Ciliary Surface Components TABLE 11. Adherence of Streptavidin-Gold Particles (SAG) to Extracted Axonemes* SAG position on axoneme

Axonemes Experiment SAG+

SAG-

Total A-B Dvnein Other Total

Trachea aa Biotin+ Biotin-

57 72

31 67

88 139

81 114

65 18

21 25

168 157

Trachea cb Biotin+ Biotin-

73 42

43 45

116 87

108 60

40 10

18 17

166 87

*Totals were from 16 micrographs for each sample. The A-B position was located at the peripheral join between A and B subfibers of an individual doublet microtubule; the dynein position was located at the outer dynein arm; other was any other location such as central microtubules. "Biotin labeled versus non-labeled axonemes with or without SAG in this experiment bordered on a significant difference ( P = ,055) by chi-square analysis. SAG position on biotin-labeled versus non-labeled axonemes was significantly different ( P < ,001). bBiotin labeled versus non-labeled axonemes with or without SAG in this experiment was significantly different ( P < .05) by chi-square analysis. Similarly, the SAG position on biotin labeled versus nonlabeled axonemes was significantly different ( P = .02).

some tracheae may have had impaired integrity and thus were penetrated by the biotin label. Two points refute this possibility: 1) ciliary movement ceases upon membrane disruption but was not diminished by incubation of tracheal epithelium in the biotin labeling solution, and 2) axonemes labeled after removal of their membranes showed a different biotin profile than axonemes labeled before isolation. The reason for variation in amount of biotin label on these high molecular weight proteins, >300 kd, is at present unknown. Inapparent differences in individual tracheae such as mucus secretion, or bacterial colonization may contribute to the variability in biotin-labeling, although the protein constituents of the isolated cilia and extracted axonemes from 5 different tracheae were reproducible. Nevertheless, there was consistent labeling of certain components in detergent-extracted axoneme fraction. This suggests close interaction of respiratory ciliary axonemes with surrounding membrane components. The nonspecific affinity of streptavidin for two components on transfers of the membrane + matrix fraction even from non-biotinylated epithelium makes it unsurprising that the streptavidin-gold particles bound to non-labeled cilia and reconstituted membrane vesicles, although in fewer numbers than to biotin labeled cilia and vesicles. The location of SAG and thus, presumably, the biotin-labeled components, did not appear to be structurally organized or specifically oriented on the organelle. The very small amount of the membrane proteins carrying the majority of biotin label relative to the total membrane protein in SDS-PAGE analysis suggests

327

that these membrane constituents occur infrequently, which is confirmed by electron microscopic observation. It is interesting that the streptavidin-gold particles bound to non-biotin-labeled intact axonemes in suspension in 1% gelatin, which prevented nonspecific interaction with separated components of these axonemes on transfers. Roth et al. [ 19891 report that a concentration of 8 nm gold particles equivalent to that of the 10 nm particles employed here gives nonspecific staining of tissue sections. However this concentration, recommended by the supplier, appeared reasonable considering the presumably greater number of potential binding sites in axonemes in suspension incubated with SAG prior to embedment. The defined, predominant site of streptavidingold adherence along the peripheral join between A and B microtubules argues a special significance. Indeed, this location has been identified previously as the site for the bridge linking microtubules to membrane, an additional ATPase [Dentler et al., 1980; Marchese-Ragona and Johnson, 19851, and for binding lectin-colloidal-gold particles [Hastie and Krantz, 19881. In addition to this apparently reactive site, streptavidin-gold particles adhered to the outer dynein arms of biotin-labeled axonemes corresponding to the detection of biotin label on the dynein high molecular weight proteins in SDSPAGE transfers of these axonemes. Other sites of biotin label on the axonemes possibly occurred too infrequently to be noted, or were not readily accessed in intact structures. In summary, a simple, rapid method of obtaining respiratory cilia with membranes in numbers adequate for further fractionation and examination has been described. Biotin labeling of the epithelium combined with this method of cilia isolation identified components accessible to the external environment which merit further study as potential reactive sites with foreign matter. Some aspects of similarity but several differences exist between mammalian and protozoan ciliary membrane constituents. The presence of biotin-labeled components in detergent-extracted axonemes indicates a close interaction of axoneme and membrane, not surprising in these organelles which respond to a variety of environmental stimuli. ACKNOWLEDGMENTS

The work presented here was performed at the Electron Microscope Facilities, Department of Anatomy, and Department of Medicine, Thomas Jefferson University, under BRS Shared Instrumentation Grants Nr. 1 SlORR01426-01A1 and Nr. 1 SlORR04910-01A1 from D.R.R., and was supported by grant R29ES04137 from the N.I.H., D.H.H.S. The technical assistance of Lisa P. Evans is gratefully acknowledged.

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Hastie et al.

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