Selective incorporation of architectural proteins into terminally differentiated molluscan gill...

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 27430M09 (1996)

Selective Incorporation of Architectural Proteins Into Terminally Differentiated Molluscan Gill Cilia

R.E. STEPHENS Department of Physiology, Boston Uniuersity School of Medicine, Boston, Massachusetts 02118

ABSTRACT Incubation of excised gills from the bay scallop Aequipecten irradians with 3H- leucine demonstrates that many ciliary structural proteins can attain a degree of labeling approaching that previously reported for sea urchin or surf clam embryos undergoing ciliary turnover or regeneration. This labeling is not a consequence of any predominant incorporation into new cilia at the meristematic growth tips of the gill since tissue regions of varying maturity incorporate label into the same proteins at similar levels, with the most mature region having the highest incorporation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorographic analysis o f isolated cilia, separated into detergent-soluble membrane/matrix and detergent-insoluble 9+2 axoneme fractions, reveals that 1) tubulin in the membrane/matrix fraction is labeled whereas tubulin in the axoneme is not; 2 ) no labeled dynein heavy chains are seen in either fraction; 3) the most heavily labeled axonemal components do not appear to any significant extent in the membranelmatrix fraction; and 4) after thermal depolymerization of the microtubules, nearly all labeled proteins reside in the insoluble ninefold ciliary remnant, the most prominent being tektin A, an integral component of outer doublet microtubules. Further fractionation of the remnant with sarkosyl-urea to produce tektin filaments demonstrates two solubility classes of tekin A, only the more soluble of which is labeled. Very similar selective architectural protein labeling patterns have been reported for steady-state cilia o f sea urchin embryos, and this may indicate a widespread turnover or exchange mechanism characteristic of cilia heretofore considered static. 0 1996 Wiley-Liss, Inc.

It is of some interest to understand the route by which newly synthesized proteins are incorporated into a growing cilium, an organelle that is uniquely compartmentalized from the protein synthetic machinery of the cytoplasm. There is little doubt that ciliary or flagellar growth must take place from the distal tip of the 9+2 axoneme (cf. Johnson and Rosenbaum, '921, requiring precursor proteins to traverse the entire length of the cilium either in a soluble form or in associa- tion with the membrane or the axoneme proper. Understanding the nature of proteins within the membrane-bound region surrounding the axoneme may provide important information for de- ciphering the mechanics of ciliogenesis, length regulation, and turnover.

The detergent-solubilized membrane/matrix fraction of scallop gill and sea urchin embryonic cilia contains tubulin as the major protein component (Stephens, '90, '91). Upon removal of detergent, this tubulin can be reconstituted into membrane vesicles of uniform buoyant density and even recycled several times without significant change in tubulin content. However, no simple post-translational modifications at the 0 1996 WILEY-LISS, INC.

amino acid level have yet been found which dis- tinguish membrane-associated tubulin from tubulin of the axoneme. In fact, it behaves as an axonemal precursor (Stephens, '92, '94a).

Acylation with palmitic or myristic acid provides one means for rendering proteins lipophilic and for determining their subcellular locations (cf. Olson et al., '85). During experimentation to determine whether scallop gill ciliary membrane-associated tubulin was fatty acid acylated, amino acid labeling controls were run to assess metabo- lism of the fatty acid to leucine. No significant hydroxylamine-labile labeling of tubulin subunits was obtained with palmitic acid, indicating that the membrane-associated tubulin was not palmi- toylated, but intense, hydroxylamine-stable labeling of numerous ciliary structural proteins was observed in the leucine control (Stephens, unpublished). These proteins represented the "architectural" elements of the 9+2 structure, in contrast

Received October 31, 1995; revision accepted January 19, 1996. Address reprint requests to R.E. Stephens, Dept. of Physiology,

Boston University School of Medicine, 80 E. Concord Street, Boston, MA 02118.

SELECTIVE INCORPORATION OF CILIARY PROTEINS 301

to the unlabeled major building blocks, tubulin and dynein. The degree of labeling approached that seen when sea urchin embryos were incubated under similar conditions of isotope level, time, temperature, and tissue mass. This was an unexpected result for a tissue whose ciliated cells are terminally differentiated.

This report explores the incorporation phenomenon with respect to gill development, tubulin synthesis, and labeled ciliary protein localization, correlating the results with recent data obtained for sea urchin and surf clam embryonic cilia (Stephens, '94a,b). A preliminary report of these basic findings has appeared in abstract form (Stephens, '89a), the implications of which were unrealized at the time.

MATERIALS AND METHODS Bay scallops (Aequipecten irradians) were ob-

tained from various Cape Cod locations and main- tained at the Marine Biological Laboratory, Woods Hole, MA, in ambient, running seawater to provide their planktonic nutritional requirements. Only fully mature, 2nd-year size class animals were used. Gills were removed by cutting the con- nective tissue holding them to the visceral mass. After excision, the gills were placed in ice-cold filtered seawater buffered with 10 mM Tris-HC1 (pH 8.0) for 15-30 min to expel mucus and adhering filtered material. The cilia can remain motile for 12-24 h under these conditions.

For scanning electron microscopy, sections of gill were fixed with 2.7% osmium tetroxide and 2.3% HgC12 in buffered seawater (10 mM Tris-HC1, pH 8.0) for 10 min (Tamm, '72). Small pieces of gill were placed in minimal volumes of seawater and instantaneously fixed by the rapid addition of >tenfold volumes of the fixing solution. The samples were then washed in distilled water, post-fured with 2% glutaraldehyde in buffered seawater, dehydrated in ethanol, critical-point dried, sputter-coated, and observed and photographed with a Nanolab-7 scanning electron microscope.

For amino acid labeling, approximately 1 g of gill tissue was incubated for 2 h at 10°C in 25 ml of seawater containing 3H-leucine at 2.5 pCi/ml (NET-460, 147 Ci/mmol, New England Nuclear, Boston, MA) with occasional agitation. No unlabeled leucine chase was used. The tissue was simply rinsed free of isotope in ice-cold, buffered seawater prior to the isolation of cilia.

Cilia were isolated by the hypertonic shock method of Linck ('73a). Gills were stirred gently for 15 rnin in ice-cold buffered seawater contain-

ing 35 g/liter of additional NaC1, filtered through Miracloth (Calbiochem, San Diego, CA), and then subjected to two cycles of 10-min differential centrifugation at 1,OOOg and 10,OOOg to sediment cell debris and cilia, respectively. The membranelmatrix fraction was solubilized by two 15-min ex- tractions with 0.5% Triton X-100 in 3 mM MgC12, 30 mM Tris-HC1 (pH 8.0), 1 mM phenylmethyl- sulfonyl fluoride, after which the demembranated 9+2 axonemes were recovered by a 10-min centrifugation at 25,OOOg.

Ciliary axonemes were fractionated by low ionic strength dialysis against >lo0 volumes of 0.1 mM EDTA, 1 mM Tris-HC1 (pH 8.01, and 0.1% 2- mercaptoethanol for 24-36 h at 4°C. This pro- cess solubilizes outer arm dynein, B-subfiber tubulin, and one central pair member (Linck, '73b). The resulting insoluble ninefold arrays of A-tubules bearing inner arms were recovered by centrifugation at 45,0008. for 15 min. The associated A-singlets were further fractionated by warm- ing at 45°C in 1 mM EDTA, 10 mM Tris-HC1 (pH 8.0), and 0.1% 2-mercaptoethanol for 15 min, yielding soluble A-subfiber tubulin and insoluble ninefold ciliary remnants (Stephens et al., '89). The latter were recovered by centrifugation at 45,OOOg for 15 min.

To produce tubulin-tektin protofilament ribbons and to fractionate them further into tubulin-free tektin filaments, ciliary remnants were subjected to increasing concentrations of urea in 0.5% sarkosyl, 1 mM EDTA, 50 mM lysine, 50 mM Tris- HC1 (pH 8.01, and 0.1% 2-mercaptoethanol. The resulting insoluble filaments, typically in 0.25- 0.5 ml of buffer, were recovered by centrifugation at 50,OOOg for 30 min (Linck and Langevin, '82; Linck and Stephens, '87). All steps were carried out in the cold.

The various fractions, adjusted to appropriate stoichiometric volumes with their respective buff- ers, were mixed with concentrated sodium dodecyl sulfate (SDS) sample buffer, boiled 2-3 min, and analyzed on 8%T/2.5%C polyacrylamide gels (1.5 mm thick x 10 cm long) with the discontinuous ionic system of Laemmli ('70), using 0.1% Sigma L-5750 sodium lauryl sulfate (Sigma Chemical Co., St. Louis, MO). The gels were stained with Coomassie Blue R-250 (Pierce Chemical Co., Rock- ford, IL) by the equilibrium method of Fairbanks et al., '71). For fluorography, the gels were treated with En3Hance (New England Nuclear, Boston, MA), dried, and exposed to pre-flashed Kodak X- Omat AR film (Eastman Kodak Co., Rochester, NY) at -80°C (Laskey and Mills, '75). To mini-

302 RX. STEPHENS

mize quenching of the 3H signal within heavily stained regions, duplicate gels were processed through the acid-alcohol stainingdestaining protocol but without the dye (Fairbanks et al., '71).

For immunoblotting, proteins resolved by SDS- polyacrylamide gel electrophoresis (PAGE) were transferred to nitrocellulose by the method of Towbin et al. ('791, modified to include 0.1% SDS in the blotting buffer. The resulting blots were processed for primary antibody binding and al- kaline phosphatase-coupled secondary antibody detection (Promega Corp., Madison, WI) accord- ing to the protocol provided by the manufacturer. A broad-range rabbit antibody raised against Lytechinus pictus (sea urchin) 51-52 kDa sperm flagellar tektin was used (Linck et al., '87). The antibody primarily detects two major scallop gill cilia bands which we have previously designated as tektins A and B, based on both their relative electrophoretic migration and their cross-reactivity with other chain-specific antibodies (Stephens et al., '89).

Stained gels, fluorograms, and western blots were quantified by computer-based video analysis (Stephens, '92) using the JAVA hardwarehoftware analysis package (Jandel Scientific, San Rafael, CA) and the principles outlined by Haselgrove and co- workers ('85). Optical density (OD) or relative re- flectance values were obtained from the 8-bit video intensity data by applying the mathematical transform, OD = log (background intensityhand intensity). For fluorograms, multiple exposures were used to insure that the film and video cam- era responses were linear; in a practical sense, this limited optical densities to <1.25.

RESULTS General clxonemal protein labeling

As noted above, preliminary results indicated that numerous scallop gill ciliary proteins were labeled after a relatively short incubation of the tissue with 3H-leucine. One plausible explanation is that the labeled proteins were derived from new cilia found chiefly on developing portions of the gill. Lamellibranch gills are crescent shaped and grow from both ends in a meristematic fashion (Ash and Stephens, '75; Good et al., '90). This is illustrated in Figure 1, providing an overview of the formation of individual gill filaments in the actively developing tip region.

To test whether most of the previously observed protein labeling was actually the result of ciliogenesis in the less mature regions of the gill,

equal weight portions of the gill's mature center, differentiated but elongating ends, and developing growth tips were excised, as illustrated sche- matically in Figure 2. These sections were incubated separately with 3H-leucine and their cilia were then isolated and detergent-extracted to yield 9+2 axonemes. The SDS-PAGE protein and 3H label pattern of equal amounts of these axonemes are illustrated in Figure 3.

Contrary to expectations based on ciliogenesis alone, typically >60% more label is incorporated into cilia from mature tissue than from either the developing growth tips or the differentiated but elongating end regions. Furthermore, the labeling pattern in all three cases is qualitatively the same, i.e., no proteins are disproportionately synthesized. In spite of being overloaded, neither the dynein heavy chains nor the a- or P-tubulin chains show detectable label. Rather, numerous minor bands representing the architectural proteins that tie the axoneme together are selectively labeled. Significant label, mainly obscured here by the heavily-stained tubulin subunits, is incorporated into proteins migrating at positions characteristic of tektins A and B, key structural components of the outer doublet microtubules (Linck and Langevin, '82; Linck and Stephens, '87) and deter- minants of the ninefold axonemal array (Stephens et al., '89). In this example, the fluorogram is of the illustrated stained gel, permitting accurate side- by-side band position comparison.

The specific activities of scallop gill cilia fall into the range of 5,700-15,600 c p d m g of protein, de- pending upon the season when the animals are collected. Incorporation is highest in the mid-win- ter months when little planktonic nutrients are available, i.e., under comparatively starved conditions, where exogenous amino acid uptake should contribute most significantly. The higher incorporation values are comparable to those obtained for regenerated cilia isolated from sea urchin or surf clam embryos labeled under very similar conditions (Stephens, '94a,b). As is also true with these embryos, the gill tissue actively takes up amino acids, in this case with a tu2 of 45 min at 10°C in an incubation containing tissue in a 1:20-25 wlv ratio with seawater. No significant differences in uptake were seen among the three tissue regions studied.

Specific labeling of membrane-associated tubulin

In addition to its role in forming the 9+2 axonemal tubules, tubulin is also the major protein

SELEC’I’IVE INCORPORATION OF CILIARY PROTEINS 303

Fig. 1. Scanning electron microscopy of the developing growth tip of a scallop (Aequipecten irradians) gdl. A- The spi- ralling, undifferentiated primordium becomes radially seg- mented, after which the segments bifurcate, growing outward to form blunt filaments. B: The filaments elongate and fold to

M

E Fig. 2. Diagram of the whole gill, showing segments that

were incubated with 3H leucine to compare ciliary protein syn-

form the double lamellae characteristic of the lamellibranch gill. C: As the filaments elongate and fold, they become pro- gressively more ciliated. D: In the growth tip, clusters of developing cilia are seen on cells adjacent to fully ciliated ones. Scale bars = 100 pm in A-C, 5 pm in D.

component of the detergent-solubilized membrane/ matrix fraction of both scallop gill and sea urchin embryonic cilia (cf. Stephens, ’90). To investigate the labeling of potentially different tubulin pools and to determine the location of various architectural proteins, the cilia were subjected to the fractionation scheme illustrated in Figure 4.

The first and second detergent-soluble membrane/matrix extracts from labeled cilia were run in parallel with axonemes, with the latter being overloaded by a stoichiometric factor of 2. The resulting gel and fluorogram are illustrated in Fig- ure 5 . To optimize the quantitation of any heavily labeled minor proteins that migrate near unlabeled major ones, the duplicate gel producing this

thesis and incorporation. G: The developing growth tip region, the very end of which was illustrated in Figure 1. E The elongating filament region where filaments must produce newly ciliated cells as they elongate. M: The mature center of the gill containing fully elongated and ciliated filaments. Scale bar = 1 cm.

RE. STEPHENS 304

G E

Stain

Fig. 3. Coomassie

M G E

3H

lue-stained SDS-polyacq -imide gel and the directly corresponding 3H fluorogram of ciliary axonemes obtained from the different regions of the scallop gill, as speci- fied in Figure 2. More labeled (but qualitatively identical) proteins are incorporated into cilia from the mature (M) region of the gill than in regions where the filaments are elongating (E) or initially growing (G). d, dynein heavy chains; a and f3, tubulin a and p chains; A-C are the relative positions expected for tektins A X .

and all of the following fiuorograms was unstained. The membrane/matrix bands corresponding to a- and P-tubulin are clearly labeled, but their axonemal tubulin counterparts show little label except for leading-edge components. Dynein heavy chain labeling is not observed in the detergent extracts or the axoneme fraction. The most heavily labeled axonemal proteins are not obvious as components in the membrane/matrix fraction. This is particularly true for band A, shown previously to correspond to the outer doublet architectural component tektin A (Stephens et al., '89). Furthermore, immunoblot analysis indepen- dently failed to detect either tektin A or tektin B in the S1 or Sz fractions (not shown).

Finally, when dialysis-solubilized B-subfiber, thermally solubilized A-subfiber, and membrane/ matrix fractions are compared quantitatively with respect to tubulin, only the membrane/matrix tu-

AXONEME

"i A H GREMNANT

4 - 1

TE KT I N S r

I - - TEKTIN FILAMENTS

Fig. 4. Flow diagram for the fractionation of cilia into detergent-soluble membrane/matrix components (Sl and Sz), the 9+2 axoneme, dialysis- and heat-solubilized B- and A-subfiber tubulins (B and A), the ninefold ciliary remnant, and sarkosyl- urea (S-U) soluble tektins and tektin-containing filaments (based on data from Linck, '73b and Stephens et al., '89).

bulin is labeled. This is illustrated in Figure 6, where dilutions containing approximately equal amounts of the respective tubulins were run in parallel. The lack of label in the A- or B-subfiber tubulin subunits proves that the leading-edge labeling seen in the tubulin of whole axonemes at much higher loadings (Figs. 3, 5 ) must be due to non-tubulin components. Conversely, when compared with Figures 3 or 5, this longer exposure of the membrane/matrix fraction further illustrates a set of labeled proteins quite distinct from those of the axoneme.

Tektin labeling in the ciliary remnant Dialysis and thermal fractionation of axonemes

produces a ninefold ciliary remnant retaining about one-fifth of the A-subfiber tubulin (Stephens et al., '89) and much of what we now know to be inner arm dynein (Linck, '73b; Stephens and Prior, '95). When the ciliary remnant is analyzed in parallel with the A- and B-subfiber tubulin, nearly all of the label remains in the ciliary remnant, as one


S, S, Ax S, S, Ax A M B M A M B M

Stain 3H Fig. 5. Stained SDS-polyacrylamide gel and a correspond-

ing but unstained 3H fluorogram of first (S1) and second (Sz) Triton X-100 extracts of gill cilia and the resulting 9+2 axoneme ( A x ) . The S1 and Sz extracts are loaded a t half the stoichiometric ratio. Both tubulin subunits in S1 are labeled, but little o r no label appears in axonemal tubulin; labeled tektin A dominates the axoneme, but it is not detected in the membrane/matrix extracts. Band designations as in Figure 3.

might surmise from the absence of label in the A and B fractions of Figure 6. Figure 7 illustrates a stoichiometrically loaded gel and corresponding fluorogram, the latter being minimally exposed to keep the major labeled band within a linear re- sponse range. Here, in contrast to Figure 6, the tubulin fractions are heavily loaded, and conse- quently about 20% of the label corresponding in migration to tektin A (and confirmed as such by antibody cross-reactivity) is now detectable beneath the a-chain in the B-subfiber tubulin fraction, a point that will be discussed below. However, it should be obvious that the major labeled band also corresponds to tektin A, and it is found chiefly in the remnant.

To localize the ciliary remnant components further, remnants were subjected to sarkosyl-urea fractionation and tektin antibody analysis. Figure 8 illustrates such a fractionation. The stained gel

Stain 3H Fig. 6. A quantitative comparison of membraneimatrix

and axonemal tubulin subunit labeling. Stained SDS-polyacrylamide gel and a corresponding but unstained 3H fluorogram with approximately equal loadings of membrane/ matrix (M), dialysis-solubilized B-subfiber (B), and thermally solubilized A-subfiber (A) tubulins. No label is detectable in the axonemal tubulin subunits a t this loading and exposure. Band designations as in Figure 3.

shows the material remaining insoluble at increasing concentrations of urea, which effectively remove a- and P-tubulin, the former being almost comi- gratory with tektin A. The blots show that the amount of tektins retained by the remnant is relatively constant after 0.5% sarkosyl extraction and that significantly more tektins A and B are not extracted until the urea concentration exceeds 3 M. Over this range, about half of either tektin A or B is solubilized, estimated densitometrically from gels or blots. Thus, there are distinct sarkosyl-urea- soluble and -insoluble classes of tektin A and B, as was also observed in both surf clam gill and sea urchin embryonic cilia (Stephens and Prior, '91; Stephens, '94a). In contrast to the extraction se- quence offlagellar tektins, where tektin C is readily removed from a relatively stable tektin A-B com- plex, ciliary tektin C is not extracted over this same range, confirming similar results obtained with surf clam 4 1 cilia (Stephens and Prior, '91).

m. STEPHENS 306

B A R

Stain

B A R

3H

Fig. 7. A quantitative comparison of label in axonemal tubulin and remnant fractions. Stained SDS-polyacrylamide gel and a corresponding but unstained 3H fluorogram of stoichiometrically loaded dialysis-solubilized B-subfiber (B), A-subfiber (A), and ciliary remnant (R) fractions. At this loading, in spite of a light exposure, some labeled architectural proteins can be detected in the B-subfiber tubulin fraction. Band designations as in Figure 3.

Remnants from labeled cilia were extracted with 0.5% sarkosyV2.5 M urea to yield these two solubility classes and then analyzed for tektin label distribution. This is illustrated in Figure 9 where again the exposure of the most heavily labeled band has been adjusted to permit accurate quantitation. In spite of the fact that about half of the tektin A or B protein is in the supernatant while the other half remains in insoluble filaments, >80% of the label in tektin A and virtually all of the lube,? in tektin B are in the soluble class. How- ever, all of the label corresponding in position to tektin C remains in the insoluble filamentous fraction, as would be expected from the above extraction results.

Here, and also in Figures 5 and 7, the specific activity of tektin A is substantially higher than that of tektin B, even though they have a similar leucine content (Linck and Stephens, '87). Al- though this may reflect differences in the rates

of synthesis or turnover, the same differential labeling was demonstrated in sea urchin embryos to be the result of protein pool size differences (Stephens, '89b, '94b).

DISCUSSION After different regions of scallop gills are incu-

bated with 3H-leucine, identical qualitative ciliary protein labeling is seen in each region but less total label is incorporated into cilia by the relatively younger tissue, even though substantial ciliogenesis must take place at the growth tip. Thus the characteristic incorporation pattern seen in the whole gill does not reflect this localized population of cells undergoing active ciliogenesis. Rather, it must reflect some other phenomenon that is, in fact, greatest in the most mature region of the gill.

The incorporation is highly selective. Numer- ous ciliary architectural proteins are labeled, but the major enzymatic and structural components, dynein and tubulin, are not. While many of the architectural proteins of scallop gill cilia are re- placed by newly synthesized ones, tektins A and B in the most stable filaments of the ninefold structure are not. This last point argues strongly against the observed labeling being due to bulk assembly or uniform turnover of the axoneme, processes wherein all of the axonemal components should be labeled, given that some new synthesis of each component has taken place. The fact that neither dynein nor tubulin were labeled in the axoneme would further support selective exchange, but there is no direct evidence for the new synthesis of either protein.

Unlabeled protein, derived from a precursor pool, may exchange or assemble and not be detected. The tubulin of the membrane-matrix fraction is, in fact, labeled. Since this tubulin may serve an axonemal precursor function, as has been argued for its counterpart in sea urchin embryos (Ste- phens, '94a), the lack of label in axonemal tubulin would imply the lack of turnover or exchange. That no dynein labeling was observed in either fraction may indicate that the translation and transport of such a large protein takes longer than the experimental protocol or simply that a pool of unlabeled, preassembled dynein exists in the cytoplasm (cf. Fok et al., '94).

Since gill cilia cannot be regenerated experimen- tally, we cannot rigorously eliminate the possibil- ity that membrane-associated tubulin is derived from a pool separate from that of the axoneme and that both axonemal tubulin and dynein are

SELECTlvE INCORPORATION OF CILIARY PROTEINS 307

Sarkosyl (%I :

Urea (M): 0 0.5 > 0 0.5 - >

0 0 0.5 1 1.5 2 2.5 3 3.5 4 0 0 1 2 3 4

a

P

Stain

Fig. 8. Sarkosyl-urea fractionation of ciliary remnants evaluated by SDS-PAGE and immunoblotting. Stain: Stoichio- metrically loaded pellet fractions. Increasing concentrations of urea strip residual a- and P-tubulin from the remnant but leave a pellet of fairly constant composition in the range of 1.5-3.0 M; evaluated densitometrically, tektin C is not extracted until the urea exceeds 3 M. Blots: Pellet and supernatant fractions,

each derived from unlabeled pools. If this were the case, and if only the architectural proteins were synthesized during ciliogenesis, the observed labeling pattern could be explained solely by ciliary growth or bulk turnover, although why this would be greater in more mature tissue is para- doxical. It would further imply that membrane- associated and axonemal tubulins are different gene products and likewise that there are two distinct pools of both tektins A and B. Nevertheless, such alternative conclusions would have important implications for protein sorting during assembly.

Very similar qualitative results have been de- scribed in the cilia of sea urchin embryos (Stephens, '94a). During steady-state turnover, the remnant- associated architectural proteins become heavily labeled, as does tubulin in the membrane/matrix

Blots

the latter a t 2.5-fold dilution. Urea initially solubilizes approximately half of the total tektins A and B, then begins to solu- bilize additional tektins a t 3 M and above. Bands previously identified as tektin A and B (Stephens et al., '89) are so marked; an additional cross-reactive band migrates between these; only very weak cross-reactivity with tektin C is found with this antibody.

fraction but not in the axoneme. Analysis after sarkosyl-urea fractionation reveals that the more soluble fraction of tektin A is preferentially labeled. Upon regeneration from labeled pools, most of the axonemal proteins become uniformly labeled, and the membrane/matrix and axonemal tubulins attain essentially identical specific activities. An important exception is tektin A, which, as judged by its initial high specific activity in comparison with tektins B and C, is made in a quanta1 or length-limiting amount as the cilium assembles (Stephens, '89b). If labeled embryos are chased with unlabeled leucine before regeneration, the more soluble class of tektin A in the regenerated cilia consists of newly synthesized but now-unlabeled protein while the insoluble class contains some previously labeled tektin A that was not in-

RE. STEPHENS 308

R S F

Stain

R S F

3H Fig. 9. Stained SDS-polyacrylamide gel and a correspond-

ing but unstained 3H fluorogram of a 0.5% sarkosyl-2.5 M urea fractionation of ciliary remnants. Most of the original remnant (R) tektin A and tektin B label appears in the sarkosyl-urea supernatant (S) fraction, leaving tektin filaments (F) bearing little label except for a band corresponding to tektin C. Desig- nations as in Figure 3.

corporated during turnover. In both sea urchin embryonic and scallop gill cilia, the newly synthesized architectural proteins, particularly the heavily labeled tektin A, are not detectable in the membrane/matrix fiaction but, rather, a small per- centage is found associated with the axoneme, dissociable by dialysis (Fig. 7, B-fraction), thermal fractionation (Stephens, '94a), or high salt treatment (Stephens, unpublished). Presumably, this fraction comprises new proteins in transit.

That both sea urchin embryonic and scallop gill cilia have two solubility classes of tektins A and B, the more soluble being exchangeable and the less soluble not, adds credence to the concept of two structural classes of tektin filaments, one an integral part of the A-B junction and the other associated with some nearby portion of the exposed A-tubule wall (Linck, '76; Linck and Lange- vin, '82). In a stable outer doublet microtubule, the latter, corresponding to the more soluble class

of tektins, may be more accessible to subunit exchange than the former, which is likely to be en- compassed by tubulin and associated junctional structures (cf. Nojima et al., '95).

Quantitatively, the level of incorporation of label into scallop gill cilia and the cilia of sea urchin embryos is also quite similar, normalized for incubation time and relative amino acid uptake. A direct comparison of ciliary protein turnover in gill epithelial cells versus embryonic blastomeres would require information on both amino acid pool size and absolute rate of protein synthesis, neither of which are currently available. However, the qualitatively and quantitatively similar selective protein exchange or turnover of integral structural components that is seen in both echi- noderm embryo and molluscan gill cilia would sug- gest that a common maintenance mechanism may be at play. How or why such an exchange of architectural elements takes place in a motile cilium remains to be determined.

ACKNOWLEDGMENTS This work was supported by USPHS grant GM

20,644 from the National Institute of General Medical Sciences. I thank Dr. Elijah W. Stommel for assistance with the scanning electron microscopy and Dr. Richard W. Linck for his generous gift of sea urchin tektin antibodies and for many helpful discussions.

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