Quantal tektin synthesis and ciliary length in sea-urchin embryos

R. E. STEPHENS

Marine Biological Laboratory, Woods Hole, MA 02543, USA

Summary

Previous work using pulse-chase labelling of em- growth in the presence of zinc ions, resulting in bryos from the sea-urchin Strongylocentrotus droe- cilia averaging nearly twice the normal 20/tin bachiensis during ciliogenesis, regeneration or length. When these embryos are pulse-chase steady-state maintenance and elongation showed labelled during ciliary growth and elongation, that a ciliary outer doublet -associated labelling of tektin A is proportional to the greater , originally termed component-20, was syn- ciliary length, as is the pool of labelled but un- thesized in a fixed or quantal amount. This sugges- incorporated tektins and other minor . De- ted that the limited synthesis of component-20 ciliated animalized and control embryos, pulse- might limit ciliary length, since the embryo has a chase labelled during their identical phases of large pool of most other ciliary components. Label- ciliary regeneration, incorporate labelled tektin A ling experiments with S. purpuratus embryos now to the same extent and have similar pools of unin- confirm quantal synthesis of component-20, while corporated proteins. The correlation of enhanced antibodies to S. purpuratus flagellar tektins tektin A synthesis with increased ciliary length and identify component-20 as the ciliary equivalent of the coincidence of tektin A synthesis with ciliary 3 elongation are observations consistent with the the flagellar 55xlO Mr tektin, tektin A. Sequential pulse—chase labelling at various times prior to hypothesis that tektin A is a ciliary length-limiting isolation of cilia proves that the high specific ac- structural element. tivity of this protein truly reflects de novo synthesis of a structurally stable protein and not rapid pro- tein turnover. Embryos may be animalized by Key words: cilia, ciliogenesis, tektin, protein synthesis.

Introduction This simple 'limiting key component' model for ciliary assembly and length control was faced with three major Earlier work showed that a ciliary outer doublet micro- reservations. First, the nature of ciliary component-20 tubule-associated protein, termed component-20, was was uncertain. Linck (1976) showed that Sarkosyl solu- synthesized de novo and in a fixed amount upon either bilization of sea-urchin sperm flagellar outer doublet normal ciliogenesis or induced ciliary regeneration in produced insoluble protofilament ribbons embryos of the cold water sea-urchin Strongylocentrotus consisting of and certain other proteins, among 3 droebachiensis (Stephens, 1977). Pulse-chase labelling them a 55 x 10 Mr protein that he termed component-20. during steady-state ciliary maintenance and elongation, However, the precise origin and nature of these flagellar which occur in later stages where cell division is minimal, proteins was unclear at that time. Furthermore, even indicated that component-20 was the major protein to be though the identification of ciliary component-20 was synthesized and fully incorporated into cilia, as judged by based both on its localization in Sarkosyl-resistant proto- its uniquely high specific activity in cilia but low specific filament ribbons and on its apparent molecular weight, activity in the residual cytoplasmic pool. These obser- extrapolation from sperm flagella of one species to vations prompted speculation that the limited synthesis blastula cilia of a different species introduced further of component-20 consequently would limit ciliary length, degrees of uncertainty. since sea-urchin embryos have a large pool of most other Later work by Linck and coworkers (Linck & ciliary protein precursors (Auclair & Siegel, 1966; Ste- Langevin, 1982; Linck et al. 1985; Amos et al. 1986) phens, 19726). Such quantal synthesis - that is, the demonstrated unequivocally that flagellar components production of limited amounts of certain critical proteins 20, 21 and 22, now known as tektins, are integral through differential expression - has been proposed structural components of outer doublet microtubules and as an important control mechanism in development that the tektins represent a new family of proteins quite (Sussman & Newell, 1972). distinct from the (Linck & Stephens, 1987). As

Journal of Cell Science 92, 403-413 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 403 to the direct extrapolation to cilia, sea-urchin ciliary degree of animalization and the relative motility of cilia were proteins that are cross-reactive with sperm flagellar tektin documented by video-enhanced Nomarski differential inter- antibodies have been described (Amos et al. 1985). ference contrast microscopy (Inoue\ 1981), using a 10X Nikon Plan DIC 025 NA objective to maximize the depth of field and An additional concern was the unusually high specific recording the data on J inch videotape. activity observed for component-20 in cilia grown during a pulse but the marked depletion of label in component- 20 from cilia regenerated after chasing with unlabelled Cilia isolation . This observation was the primary basis for At the appropriate stage, the embryos were recovered by gentle proposing limiting or quantal synthesis, but one could manual centrifugation, washed several times with Millipore- theoretically explain the observation if the protein had a filtered sea-water, and deciliated by a 2-min treatment with double tonicity sea-water (Auclair & Siegel, 1966; Stephens, half-life somewhat shorter than the total time for a single 19726) at 4°C. This method has been described in detail in a round of ciliary growth. The question of rapid protein recent review (Stephens, 1986). Resuspension of the embryos turnover had not been addressed rigorously. Although it in normal tonicity sea-water permitted full regeneration of cilia could be argued that any protein presumably so integrally in 8h at 8°C (Stephens, 1977). The length distribution for associated with outer doublet microtubules should not be populations of isolated cilia was determined by minor modifi- subject to turnover within the assembled , rapid cations of methods outlined by Burns (1973). Protein determi- turnover of newly synthesized component-20, prior to nation was by the method of Lowry et al. (1951). assembly, or metabolism within an unassembled pool, prior to regeneration, would be consistent with the Labelling procedures and analysis observations. Identical cultures of developing 5. droebachiensis embryos Finally, even if this ciliary protein is the equivalent of were pulse-labelled, at various points in development, for 4h I4 3 flagellar component-20, and even if it is synthesized with 2-5^Cimr' of [ C]- or [ H]leucine and chased for 4h quantally, none of the earlier data could correlate the with a 1000-fold excess of unlabelled leucine, essentially as amount of component-20 synthesized with the total mass described (Stephens, 1977). At a time dictated by the exper- imental protocol, the virgin cilia, labelled during the pulse, of cilia assembled. If component-20 was localized in one were isolated. The now-deciliated embryos were returned to region of the cilium, quantal synthesis could simply normal sea-water and were allowed to regenerate cilia, utilizing reflect the number of cilia made rather than their length. protein pools labelled during the first generation. The regener- However, it is now known that the three flagellar tektins ated cilia, in turn, were isolated for comparison with the virgin are distributed uniformly along the entire length of outer cilia to evaluate labelled pool depletion. doublet microtubules (Linck et al. 1985; Amos et al. Parallel cultures of embryos, normal and zinc-animalized, 1986; Steffen & Linck, 1988) and thus one should expect were similarly pulse-chase labelled at various points in develop- a length correlation if quantal tektin synthesis during ment and their virgin and regenerated cilia obtained as above. ciliogenesis is a length-limiting factor. In addition, parallel cultures of embryos, normal and anima- lized, were initially deciliated, pulse-chase labelled for specific This current report establishes the identity of the times during regeneration, deciliated again to provide first quantal ciliary component-20, addresses the question of regeneration cilia, and then allowed to produce second-regener- its turnover, and correlates its quantal synthesis with ation cilia, removed for analysis by a third deciliation. Further ciliary length. A brief report of these findings has details of pulse-chase times with respect to the kinetics of appeared in abstract form (Stephens, 1987). regeneration are given in Results. For comparative analysis, virgin and regenerated (or first and second regeneration) cilia were demembranated by extraction Materials and methods for 5min with 0-25% Triton X-100 in 3mM-MgCl2 buffered with30mM-Tris-HCl, pH80, then spun at 25 000 g for lOmin. Synchronized embryos The resultant 9+2 axoneme pellets were analysed by Eggs from the sea-urchin Strongylocentmtus droebachiensis SDS-PAGE on 8% slab gels (0-15cm thickXlOcm long), were fertilized, washed thoroughly with Millipore-filtered sea according to the method of Laemmli (1970). For protein water, subdivided into equal portions in 250 ml siliconized quantification, the gels were stained with either Coomassie Blue beakers, each containing 1 ml of eggs in 25 ml of sea water, and (Serva) or Acid Fast Green FCF (Gorovsky et al. 1970), using grown at 8°C in a temperature-controlled bath. Additional equilibrium staining (Fairbanks et al. 1971). For fluorography, details of gamete procurement, culture conditions, and time- the gels were treated with En3Hance (New England Nuclear), tables of development have been published (Stephens, 1972a). dried, and exposed to pre-flashed Kodak X-Omat AR film, Animalized embryos were produced basically as described by normally for 1-4 days at —80°C. Using an Ortec 5310 photon- Nemer et al. (1985) for the related sea-urchin 5. purpuratus. counting densitometer, relative specific activity was determined This simply involved the addition of ZnCl2 to a final concen- by densitometry of the stained gel and its equivalent fluoro- tration of 05 mM in the sea-water culture medium immediately graph, taking care to use multiple sample loadings and/or film after fertilization. The treatment had no measurable effect upon exposures to remain within the linear limits of these methods. the normal timing of development up to hatching, which was For labelling of S. purpuratus cilia during regeneration, slightly delayed even though the embryos themselves appeared embryo culture was carried out as outlined above, but growth equivalent to the untreated control in terms of cell number and was at 14°C. Labelling was with [ CJleucine, applied immedi- onset of ciliogenesis. The animalized embryos had the expected ately after deciliation as a 2-h pulse, followed by a 2-h chase. longer cilia but were unable to rotate within their more resistant Another deciliation, regrowth for 4h, and a final deciliation fertilization envelopes. Gentle centrifugation and resuspension yielded first- and second-regeneration cilia. Subfractionation of caused rapid dispersal of the mechanically fragile envelopes but the axonemes by low-ionic strength dialysis and extraction with only after the time point coincident with normal hatching. The 0-5 % Sarkosyl were carried out exactly as described (Stephens,

404 R. E. Stephens 1977), as was the analysis by direct autoradiography and densitometry.

Western blotting For absolute identification of ciliary tektins with their flagellar counterparts, one-dimensional gels, as above, or two- dimensional gels, as described by O'Farrell (1975), were probed with affinity-purified polyclonal antibodies raised against the 3 47, 51 and 55(XlO )Mr tektins from S. purpuratus sperm flagella (Linck et al. 1987). The gels were blotted to nitrocellu- lose with a Tris-glycine transfer buffer containing 20 % meth- 20 anol and 0-1% SDS (Burnette, 1981) at 60 V for 1 h. The equivalent ciliary tektins were localized with flagellar primary antibodies, using an alkaline phosphatase-labelled secondary antibody (Promega Biotec). Tubulin chains were localized using a polyclonal antibody to sea-urchin egg tubulin (Poly- sciences no. 17870) or a monoclonal antibody to rat brain /3-tubulin (1CN no. 63-781). The developed blots were thoroughly dried and subjected to 14C direct autoradiography with pre-flashed Kodak X-Omat AR film. Replicate experimen- tal and control blots were aligned with the corresponding autoradiograms to identify the components in question.

Results

Ciliary component-20 is antigenically equivalent to flagellar tektin A The original identification of ciliary component-20 in S. clroebachiensis was based on its electrophoretic co-mi- gration with the presumptive flagellar outer doublet counterpart from this same species. That analysis utilized Sarkosyl-resistant ciliary protofilament ribbons com- posed mainly of the two tubulin subunits and three Fig. 1. SDS-PAGE analysis of Sarkosyl-resistant proteins in the tubulin region, which co-migrated with protofilament ribbons derived from S. purpuratus embryonic 14 sperm flagellar component 20, 21 and 22, these being cilia, labelled with [ C]leucine during regeneration and then defined only by their relative position (cf. Linck, 1976). regenerated a second time. Vertical bar, 0-75 O.D. unit for Quantal synthesis of a bona fide component-20 had not trace a, and IOO.D. unit for traces b,c; horizontal bar, 1-0 cm. Trace a, densitometer trace of a Coomassie Blue- been demonstrated in S. purpuratus, the species from stained gel; ribbon-associated components are denoted by which most of the current tektin biochemical data are numbers as defined by Linck (1976); T, unresolved tubulin derived, and one could argue that the protein in question 3 subunits. Note that a minor polypeptide (about 53xl0 /17r) is not one of the tektins. The presence of a minor occurs between components 20 and 21. Trace b, densitometer 3 53xl0 7V/r protein in tektin filament preparations from trace of the autoradiogram derived from a, indicating proteins 5. purpuratus (Linck et al. 1987), further complicated labelled during the first regeneration. Component-20 is the by migration differences among tektins from different most heavily labelled major protein but certain trace species (Steffen & Linck, 1988), leaves open to question components (asterisks) are disproportionately labelled as well. the identification of a particular tektin by electrophoretic Trace c, densitometer trace of the equivalent second (cold) mobility alone. regeneration, where the label reflects proteins synthesized during the first regeneration but not incorporated until the To address the questions of quantal synthesis and second. Note the depletion of label in component-20 and the species equivalence, mesenchyme blastula embryos of S. askerisk-denoted proteins from trace b and that more label purpuratus were deciliated and pulse-chase labelled appears in the tubulin subunits. during ciliary regeneration, deciliated again, and allowed to re-grow cilia from previously labelled pools. The resulting first- and second-regeneration cilia were frac- labelled protein in the second regeneration (78 % de- tionated to Sarkosyl-resistant protofilament ribbons and crease, based on peak heights). The other components, these were analysed by SDS-PAGE and autogradiogra- like most other ciliary proteins, decrease somewhat phy, the results of which are illustrated in Fig. 1. (20—25%), reflecting utilization from relatively large As is true in S. clroebachiensis cilia, a protofilament- labelled pools (cf. Stephens, 1977). Unlike 5. clroeba- associated protein that migrates just ahead of ^-tubulin, chiensis cilia, there is an additional minor protein be- corresponding to flagellar component-20, incorporates a tween the highly labelled component and component-21, 3 very high degree of label during the initial regeneration, most likely corresponding to the 53xl0 iV/r minor com- but this labelled protein, synthesized and incorporated ponent of flagellar tektin filaments (Linck et al. 1987). during the first regeneration, is greatly diluted by un- This protein is not preferentially labelled during the

Quantal tektin synthesis and ciliary length 405 Fig. 2. Two-dimensional immunoblot analysis of pulse-chase labelled first- and second-regeneration ciliary axonemes to identify quantal component-20. A. Autoradiogram of a two- dimensional gel blot of regenerating cilia pulse-labelled with [14C]leucine for 4h after 2h of initial growth, chased for 2h, then isolated. The a- and /3-tubulin chains are indicated, as is 3 a S5xlO Mr protein (component-20) that is heavily labelled during ciliary growth. B. Autoradiogram of a two- dimensional gel blot of second-regeneration cilia; the numerical designations indicate the proteins identified by immunoblotting. Isoelectric points (pi) are indicated. Anti- 55: horizontally aligned portion of an immunoblot from a replicate two-dimensional gel demonstrating cross-reactivity .55 of component-20 with an antibody to S. purpuratus sperm 3 flagellar 55xl0 iWr tektin. Note additional, more-acidic cross- reactive proteins of the same molecular weight. Anti-51: 3 same, using an anti-51 XlO Mr tektin. Anti-47: same, using 3 an anti-47Xl0 yWr tektin. Note additional, weaker cross- 3 reactivity with the 55xlO Mr tektin.

it actually shows a higher degree of labelling in second- B regeneration-cilia (cf. Stephens, 1977). Immunoblotting of one-dimensional gels of pulse- chase labelled virgin and regenerated cilia from 5. droebachiensis embryos, utilizing S. purpuratus flagellar tektin antibodies, consistently demonstrates that the heavily labelled ciliary component-20 precisely co-mi- grates with (or is) a protein antigenically equivalent to the 3 flagellar 55xlO Mr tektin (data not shown). However, proximity of component-20 to /3-tubulin makes precise a alignment of the gel, the blot and the autoradiogram .55 difficult, particularly in the case of S. purpuratus where 3 there is the additional 53xlO A/r protein component flanked by components 20 and 21, as noted above. 47 To demonstrate equivalence rigorously, two-dimen- sional immunoblotting, as was used by Linck and co- workers (1987) to characterize flagellar tektin antigens Pi 7 6-5 6 5-5 and antibodies, was applied to regenerating cilia. Fig. 2 in illustrates autoradiograms of mesenchyme blastula ciliary in axonemes isolated after being pulse-chase labelled mid- way through an experimentally induced regeneration (Fig. 2A) and isolated again after regrowth from the resulting labelled pool (Fig. 2B). This procedure permits moderate labelling of all axonemal proteins for reference purposes while still permitting observation of a >70% i reduction in the label of component-20 upon a second i regeneration (cf. Stephens, 1977, and below). Sections of immunoblots from replicate two-dimensional gels (Fig. 2: anti-55, -51, -47) identify three minor com- ponents in the autoradiograms as antigenically equivalent to the three flagellar tektins. Not only do these corre- c spond in terms of molecular weight and isoelectric point < to the S. purpuratus flagellar tektins, but even the 3 multiplicity of 47 and 55 X10 A/r isoforms and the cross- 3 initial regeneration. Like 5. droebachiensis cilia, label in reactivity of anti-47 with the 55 X 10 Mr tektin equivalent one higher and several lower molecular weight proteins is are quite similar, in spite of derivation from a different also depleted (Fig. 1, asterisks) but in that species these organelle and species (cf. Amos et al. 1985; Linck et al. proteins were less tightly associated with the insoluble 1987). Therefore, S. droebachiensis embryonic ciliary protofilament ribbon and were found mainly in the component-20 is antigenically equivalent to the 3 A-tubule fraction. As in S. droebachiensis, tubulin 55xl0 iV/r sperm flagellar tektin of S: purpuratus, now synthesized during the first regeneration is not fully known as tektin A, on the basis of this two-dimensional utilized from the pool until the second and, consequently, analysis.

406 R. E. Stephens Quantitative labelling experiments at mutiple time Hours: +46 50 54 58 70 points were not attempted using the two-dimensional gel/immunoblotting/autoradiography approach because a a the methods are not inherently quantitative, owing to the h«H I I I I I I I I M» limited solubility of tektins in urea (cf. Linck & Ste- Mesenchyme blastula phens, 1987) and the variable loss of tektins in the first b b' dimension or during blotting (cf. Linck et al. 1987). 'II I I I I I I I II I* However, both one- and two-dimensional analyses were Invagination occasionally employed to identify unequivocally some of the nearly comigrating proteins described below. Regard- less of issues of absolute resolution, the one-dimensional Mid-gastrula semi-quantitative approach taken here is intended to • pulse yield strictly parallel experimental conditions for direct i chase visual and densitometric comparison. * deciliation H- growth/regeneration

Ciliary tektin A is metabolically stable Fig. 3. Schematic diagram of a sequential After the initial generation of cilia by a sea-urchin embryo pulse-chase/simultaneous cilia isolation and regeneration and the slowing of ectodermal cell division at the late protocol. The times indicated are in hours after fertilization; blastula stage, existing cilia elongate progressively toward letter designations refer to samples analysed in Fig. 4. the animal pole, evidently in equilibrium or a steady state with respect to unassembled ciliary precursors. It was shown previously that when late gastrula embryos were pulse-chase labelled without removing their cilia and Virgin Regenerate these pre-existing steady-state cilia were then isolated a b c a b c a' b' C a' b' c' much later, component-20 (now tektin A) was the maj labelled protein. The subsequently regenerated cilia we 1 nearly devoid of labelled tektin A but showed hea' labelling of tubulin, presumably synthesized during tl earlier pulse-chase but not incorporated into steady-sta cilia until deciliation-induced regeneration (Stepher 1977). Since that observation involved only one tin point, relatively late in development, the possibility rapid turnover as an explanation for the high speci: activity of tektin A in either that or related experimer could not be eliminated rigorously. Consequently, ; experiment was designed wherein fully ciliated la blastula to mid-gastrula embryos were labelled at thr progressively earlier points prior to a common time cilia isolation, at which point all were allowed to regent ate cilia and these regenerates were analysed with respe to the virgin steady-state cilia. This sequential labellii — w W X protocol is illustrated in Fig. 3, while the resultii X stained gels and corresponding autoradiograms are shov Y Y in Fig. 4. The low level of incorporation of labelled tubul subunits during such a steady-state condition perm: ready evaluation of tektin A and B labelling. Upon regeneration, however, the tubulin synthesized but not Stain Fluorogram Stain utilized during the pulse-chase appears in the regener- ated cilia and, consequently, tektin A must be estimated Fig. 4. SDS-PAGE analysis of ciliary axonemes pulse-chase as a shoulder on ^-tubulin. Two-dimensional gel analysis labelled with [3H]leucine at various time points prior to reveals that a- and /3-tubulin have essentially the same isolation. Fluorograms of virgin and regenerated cilia are specific activity, a reflection of their equivalent leucine flanked by the respective Coomassie Blue-stained gels from content (cf. Stephens, 1978). Therefore, the amount of which they were derived. Time points a-c and a'-c' correspond to virgin and regenerated ciliary axonemes from label in /3-tubulin can be approximated by measuring parallel cultures that were pulse-chase labelled as specified in O'-tubulin, which migrates unflanked by labelled minor Fig. 3. The a- and /3-tubulin chains and tektins A, B, C are proteins. designated accordingly; V, W, X, Y denote quantally As was true in labelled late gastrula embryos (Ste- synthesized, non-protofilament ribbon associated proteins; Z phens, 1977), tektin A is the major labelled protein designates a mobility reference protein that is partially incorporated into ciliary axonemes of earlier stages. depleted under different experimental conditions; D denotes Contrary to expectations based on rapid protein turn- heavy chains.

Quantal tektin synthesis and ciliary length 407 over, the relative degree of tektin A labelling is actually highest in the earliest labelled sample (mesenchyme blastula), a fact that may reflect the greater ciliary elongation known to occur at that stage (Fig. 4: virgin lane a versus lanes b,c). The labelled tektin A is mainly replaced with the unlabelled form in the regenerated cilia, but the equality of tektin A label in the pool at the three successive time points (Fig. 4: regenerates, lanes a'-c') demonstrates that this residual tektin A must be as metabolically stable as that incorporated into the axon- eme. The ratio of tektin A label in steady-state versus regenerated cilia is about 4:1, a ratio seen in these and a number of earlier experiments that may reflect an equilib- rium between assembled ciliary tektin A and its cytoplas- mic pool. In addition to tektin A, tektin B and at least four other proteins (denoted V, W, X (a doublet) and Y in Fig. 4) are depleted upon regeneration. The quantal components here designated as V, W and X were observed earlier and were shown by dialysis and deter- gent fractionation not to be specifically associated with the Sarkosyl-resistant protofilament ribbon but could be solubilized with the bulk of the A-tubule (Stephens, 1977). The near-constancy of label in most other axon- emal components in equivalently but more distantly labelled samples (cf. fluorogram lanes a and b or a' and b' in Fig. 4) would suggest that both the virgin axonemes and the protein pools from which regenerated axonemes arise must be relatively stable metabolically. Independent of arguments based on depletion of labelled tektin A upon regeneration, it should be pointed out that the initial differential labelling of tektin A (versus tektins B or C) is a true measure of synthetic differences among the tektins and not a reflection of any unique compositional differences, since the leucine con- tent of the three tektins is nearly identical (Linck & Stephens, 1987). Furthermore, the leucine content of the tektins (about 9 mole %) is not unusually high with respect to other proteins, for example, tubulin (about 7 mole %), and thus an anomalously high leucine content cannot account for the relatively high degree of tektin A labelling. Fig. 5. Representative control (A) and zinc-animalized (B) In particular, tubulin (and also many lesser com- sea-urchin embryos demonstrating the marked degree of ciliary elongation at the early gastrula stage, induced by the ponents) is more heavily labelled in regenerated cilia, presence of O'SmM-zinc ions. Video-enhanced, stopped-frame confirming earlier results obtained at a different stage and Nomarski differential interference contrast micrographs interpreted as delayed utilization of a presynthesized obtained from a videotape recording of living embryos; pin, protein pool (cf. Stephens, 1977). This is especially primary mesenchyme. Bar, lOOjUm. evident at the third time point where the cilia have only the length of the pulse in which to incorporate newly regeneration, most axonemal components, with the im- labelled proteins (Fig. 4, fluorogram lanes c and c'). portant exception of tektin A, contain as much or even Here, with the notable exception of tektin A, the newly more label in the cold second regeneration. This is synthesized proteins, reflected as incorporation into re- particularly obvious in the tubulin subunits, whose generated cilia, are more heavily labelled. This is most synthesis is known to be stimulated by deciliation (cf. obvious in the dynein heavy chains, prominent at the top Stephens, 1977; Merlino et al. 1978). of the gel, where label appears almost exclusively in the second (cold) regeneration, a likely consequence of the Zinc-animalization approximately doubles average time needed to complete the translation of such unusually ciliary length large polypeptides. Zinc treatment of S. droebachiensis embryos causes Delayed utilization similar to that noted above was maximal animalization and the suspension of develop- created intentionally in the labelling scheme employed ment at the early gastrula stage, essentially as described for immunological identification in Fig. 2. Because the by Nemer et al. (1985) for S. purpu ratus. The resulting pulse was applied mid-way through an induced ciliary embryos have strikingly long cilia (Fig. 5), so long in fact

408 R. E. Stephens that the embryos are unable to swim properly and remain on the bottom of the culture dish, rotating in place by means of their quite motile, relatively short cilia at the 40 vegetal pole. The apical tuft cilia approach 80/im in length and are nearly immotile, while the cilia that cover much of the animalized embryo have a fairly uniform gradient of length, decreasing vegetally, there approach- ing the 20 [im length characteristic of the untreated 30 controls. The longer cilia slowly beat sinousoidally but as ciliary length becomes progressively shorter the mode of S beat becomes more rapid and normal. Si Direct length measurements of isolated cilia from g 20 control and zinc-animalized embryos indicate that the animalized embryos are able to produce approximately twice the mass of cilia as the controls, with no difference in cell number, in basic agreement with workers who used trypsin treatment to animalize other sea-urchin 10 species (Riederer-Henderson & Rosenbaum, 1979; Burns, 1979). The mean length values are 19-6 ± 3-0^m (S.D., n = 250) for control embryos and 35'l±6-5^tm (S.D., n = 125) for animalized embryos at the early gastrula stage. Direct protein measurements from these 2 4 6 8 10 12 same populations give a ratio of l:l"82 + 0"ll (S.D., Time (h) n = 6), in excellent agreement with length measure- Fig. 6. Kinetics of ciliary regeneration for control (•) and ments, where the summation of observed lengths for an zinc-animalized (O) sea-urchin embryos at the early gastrula equivalent number of cilia gives a mass ratio of 1:1-79. stage. Points and error bars ( + S.D.) at the origin indicate the Both estimates are probably low, however, since the very ciliary length distribution of the original populations before long apical tuft cilia of the animalized embryos are deciliation. frequently lost when the embryos are washed prior to deciliation. Furthermore, direct observations of control and animalized embryos would indicate that topologically Hours: +24 30 36 equivalent cilia are often two to four times longer (cf. 42 48 54 60 a/d a'/d' In the case of regenerating cilia from control and zinc- *1 I I I I I I IIl« animalized embryos, the initial phase of ciliary regener- ation takes place at essentially the same rate (Fig. 6). The growth of cilia on animalized embryos continues beyond b/e b'/e' MINIMI* the control length, at first nearly linearly, then at a decelerating rate until the animalized population achieves an average length approximating that of the population c/f c'/f before deciliation. This parallel kinetic behaviour is t I M I II M * Mesenchyme blastula basically the same as that described by Burns (1979) for regeneration in trypsin-animalized tropical sea-urchin embryos and by Harlow & Nemer (1987) for zinc- g pulse a-b-c, zinc animalized gf chase d-e-f, control animalized S. purpuratus embryos, although the absol- • deciliation ute rates differ. H- growth/regeneration

Synthesis of tektin A correlates with ciliary length Fig. 7. Schematic diagram of parallel pulse-chase labelling If the synthesis of tektin A is quantal but unrelated to of zinc-animalized and control embryos during ciliogenesis ciliary length, then an embryo with twice the mean length and growth. Times are hours after fertilization; letter of cilia should yield a population of cilia whose tektin A designations refer to samples analysed in Fig. 8. will contain about half the relative amount of label as cilia from a control embryo. To test this, control and anima- For convenience in direct visual comparison of label- lized embryos were pulse-chase labelled at three success- ling, axonemes of virgin cilia from animalized embryos ive points in development: during ciliogenesis, at hatch- (Fig. 8: lanes a, b, c), which yield approximately twice ing and early in gastrulation. The embryos were the amount of protein as the controls, were diluted 1:1 to deciliated and then allowed to regenerate cilia. This yield amounts of protein approximately equal to those of parallel labelling protocol is outlined in Fig. 7. The the controls (Fig. 8: lanes d, e, f). Axonemes of the axonemes of virgin and regenerated cilia from animalized corresponding regenerated cilia from animalized and and control embryos were then compared by quantitative control embryos were loaded in a 1:2 protein ratio such SDS-PAGE and fluorography (Fig. 8). that approximately equal amounts of radioactivity were

Quantal tektin synthesis and ciliary length 409 Stain Fluorogram Animal Control Animal Control a b c d e f a b c d e f

c

a A B

w X Y

Fig. 8. SDS-PAGE analysis of virgin and regenerated ciliary axonemes from zinc-animalized (lanes a-b) and control (lanes d-f) embryos labelled 1 with [3H]leucine during ciliogenesis

410 R. E. Stephens 1 st Regenerate 2nd Regenerate c' z' c' z'

9UJU

Fig. 9. SDS-PAGE analysis of first- and second-regeneration ciliary axonemes from control and zinc-animalized embryos, deciliated first and then labelled with [3H]leucine during their 6-h equivalent linear phases of ciliary a growth, c, control; z, zinc-animalized first- regeneration axonemes; primed designations are /H second-regeneration axonemes. The slightly > —Q— greater amount of axonemes from zinc- animalized embryos in the first regeneration reflects the fact that such cilia grow longer than W those of the control during the 2-h chase period X (cf. Fig. 6). In the second regeneration, the two populations were loaded at equivalent protein amounts. Coomassie Blue staining. Stain Fluorogram Stain Protein designations as in Fig. 4. axonemes of regenerated cilia from animalized embryos cilia were then isolated. Since the growth rates are the (Fig. 8: lanes a', b', c'), loaded at half the protein same during the labelling period, the two populations amount, the amount of label remaining in tektin A (and should each have cilia containing a similar length of most other labelled proteins including tubulin) at least labelled axoneme. However, the final axonemal length equals that of the control (Fig. 8: lanes d', e', f), will be about 40% more for cilia from animalized indicating that the relative specific activities of proteins embryos since these cilia continue to grow longer during remaining in the unassembled pool from animalized the chase period. The direct labelling of such cilia from embryos must be about twice those of the control. The normal and animalized embryos regenerating in parallel, other labelled minor components are also depleted upon and the residual labelling of their equivalent second regeneration, but few to the same extent as tektin A. regenerates (reflecting proteins synthesized during the Again, exceptions are the four quantal proteins desig- first regeneration but not able to incorporate into final- nated V, W, X and Y, plus another designated Z that is length cilia), are compared in Fig. 9. also labelled quantally under this protocol. These five In this analysis, ultrapure SDS, a 12 cm long gel and a polypeptides roughly correspond in electrophoretic mo- lower voltage gradient were used to increase the degree of bility to the quantally synthesized, non-tektin polypep- separation between tektins A and B. At sufficiently low tides that remain associated with S. purpuratus protofila- protein loading, these tektins flank (rather than run ahead ment ribbons (Fig. 1, asterisks). of or with) the /S-tubulin chain. Demonstrable by densi- tometry but visually confounded by the marked increase in /3-tubulin labelling in animalized embryos, the amount Tektin A synthesis and ciliary elongation are coincident of label incorporated into tektin A in axonemes from It was noted above that the initial rates of ciliary regenerating animalized embryos is indistinguishable elongation are essentially the same for control and from that of the control (Fig. 9: first regenerate, lane c animalized embryos; the latter simply continue to grow versus lane z), in spite of the fact that label was present their cilia to greater lengths (cf. Fig. 6). If tektin A during most of the control regeneration but during only synthesis and incorporation into elongating cilia are about two thirds of the animalized regeneration (cf. coincident events, cilia regenerating from control and Fig. 6). The same is true of the labelled tektin A animalized embryos during their initially coincident remaining in the respective unincorporated pools (Fig. 9: growth phases should incorporate labelled tektin A to the second regenerate, c' versus z'), in spite of the fact that same extent and, after deciliation, their residual labelled cilia from the animalized embryos had not achieved their cytoplasmic pools of tektin A should be equal. To test full length in the first regeneration. These two obser- this, the two kinds of embryo were deciliated simul- vations demonstrate that tektin A synthesis and ciliary taneously, pulse-labelled for 6h during their coincident, elongation (assembly) must be coincident events. The near-linear phases of growth and chased for 2 h, and their synthesis and incorporation of other minor axonemal

Quantal tektin synthesis and ciliary length 411 proteins during the first regeneration is similar but not zinc-animalized (but non-deciliated) embryos illustrated identical in control versus animalized embryos (see in Fig. 8. However, as noted above, the zinc-animalized below), and most minor proteins show a relatively slight regenerates also have nearly equivalent tubulin labelling, depletion (in comparison with tektin A or the proteins V, despite being loaded at half-concentration, a result con- W, X and Y) upon second regeneration, again reflecting sistent with the zinc-animalized embryos having a pro- relatively large and, in this case, equally labelled pools. portionately larger labelled tubulin pool that was not fully This kinetic regeneration experiment, wherein label is utilized until regeneration. applied only during the coincident growth phases of cilia from deciliated control and zinc-animalized embryos, yields nearly equivalent degrees of tektin A labelling in Discussion the assembled axonemes and unassembled cytoplasmic pools of both populations, as expected for coincident Component-20, the single most heavily labelled axonemal tektin A synthesis and ciliary elongation. This result is in protein during sea-urchin ciliogenesis, steady-state main- direct contrast but complementary to that obtained by tenance and induced regeneration, is the ciliary equival- labelling during natural growth and elongation, as de- ent of sperm flagellar tektin A, an integral component of scribed above. There, the amount of tektin A synthesized outer doublet microtubules. Quantally synthesized de and incorporated into axonemes is proportional to the novo and incorporated almost entirely into nascent cilia, final ciliary length. The pool labelling in animalized tektin A is metabolically stable, both in the assembled embryos is also greater than the control by a factor of axoneme and in the cytoplasmic pool. Synthesis of tektin nearly 2, suggesting that an equilibrium may determine A and other architectural ciliary proteins is coincident the twofold greater difference in ciliary length. with and proportional to the growth of the ciliary The design of the experiment illustrated in Fig. 9 is axoneme. such that any newly synthesized architectural proteins These observations are consistent with the original that might be required for a larger pool in the zinc- 'limiting key component' model for ciliary assembly, animalized embryos would be incorporated into the first- wherein component-20 (now ciliary tektin A) was pro- regeneration cilia, since these cilia grew longer than the posed as a length-limiting protein that must co-assemble controls during the chase. This may explain the relatively with the ciliary outer doublets (Stephens, 1977): In the heavier labelling of many minor components in the first- decade since that proposal was made, the tektins have regeneration cilia from the zinc-animalized embryos but been established as unique structural proteins within equalization with the control after the second regener- outer doublet microtubules (Linck & Langevin, 1982; ation, where the two populations of cilia were allowed to Linck et al. 1985; Linck & Stephens, 1987), extending attain the same length. the full length of the A-tubule (Steffen & Linck, 1988), and forming, with tubulin, the junctional protofilament Stimulation of tubulin synthesis by deciliation and domain to which radial spokes and interdoublet linkages animalization connect to give rise to the basic 9+2 structure (Stephens Deciliation typically stimulates tubulin synthesis at a etal. 1989). transcriptional level (cf. Merlino et al. 1978: Gong & Tektin A is not, of course, the only protein whose Brandhorst, 1987). One major difference between the synthesis appears to be quantal. Other proteins associated deciliated control and zinc-animalized embryos is the with the A-tubule and protofilament ribbon behave in markedly higher incorporation of label into tubulin much the same manner. If anything, this multiplicity subunits of the first-regeneration cilia of animalized strengthens the limiting key component hypothesis, since embryos, compared with those of the non-animalized many of these are mutually associating proteins, as control, and the continued higher specific activity of the evidenced by their retention in S. puipuratus protofila- tubulin subunits in the second-regeneration cilia of the ment ribbons (Fig. 1, asterisks) and by the fact that animalized embryos. Apparent stimulation is also seen nearly all architectural proteins remain associated as a for several other axonemal proteins (e.g. Fig. 9: tektins ninefold symmetric ciliary skeleton after removal of most B, C and proteins W, X and Y, although barely in V) but of the tubulin by thermal fractionation (Stephens et al. none of these continue to show a higher degree of labelling 1989). However, quantal synthesis of tektin A is still the in the animalized embryos upon second regeneration (see most likely single limiting event, since this tektin, in a 1: 1 possible explanation above). With the important excep- molar ratio with tektin B, forms the most stable complex tion of the two tubulin subunits, the second-regeneration within the axoneme, unequivocally extending from base fluorogram lanes of axonemes from control and zinc- to tip. animalized embryos in Fig. 9 are virtually indistinguish- Nemer (1986) has shown that the transcription of able. The enhancement of tubulin synthesis by zinc- certain ectodermal is markedly enhanced as a result animalization is in agreement with the observations of of zinc-animalization. It is evident from the above data Harlow & Nemer (1987) who noted that, while the that virtually all of the minor ciliary proteins must induction of /J-tubulin mRNA by deciliation is transcrip- undergo enhanced expression in order to have equal (or tionally regulated in the normal embryo, it is additionally greater) specific activities in cilia from animalized em- post-transcriptionally regulated in the zinc-animalized bryos. This leaves unanswered the tantalizing question of embryo. Note that there is no difference in axonemal whether those ectodermal genes whose transcription is tubulin subunit labelling in virgin cilia from control and enhanced by animalization represent the coordinately

412 R. E. Stephens controlled minor ciliary proteins, including the quantally of minor components and a-tubulin from specific regions of the A- synthesized tektin A, that become active at and presum- tubule.J. Cell Sci. 20, 405-439. LINCK, R. W., AMOS, L. A. & AMOS, W. B. (1985). Localization of ably trigger ciliogenesis (cf. Stephens, 1977). In support tektin filaments in microtubules of sea urchin sperm flagella by of this, preliminary evidence indicates that tektin A immunoelectron microscopy. J. Cell Biol. 100, 126-135. mRNA levels markedly increase at ciliogenesis and are LINCK, R. W., GOGGIN, M. J., NORRANDER, J. M. & STEFFEN, W. further enhanced as a consequence of zinc-animalization (1987). Characterization of antibodies as probes for structural and (Norrander et al. 1988). Such differential gene ex- biochemical studies of tektins from ciliary and flagellar microtubules. J. Cell Sci. 88, 453-466. pression is precisely what would be required under the LINCK, R. W. & LANGEVIN, G. L. (1982). Structure and chemical limiting key component hypothesis for ciliary assembly composition of insoluble filamentous components of sperm flagellar (Stephens, 1977), thus rendering ciliogenesis in sea- microtubules. J. Cell Sci. 58, 1-22. urchin embryos a prime example of a developmental LINCK, R. W. & STEPHENS, R. E. (1987). Biochemical event that is under 'quantal control' in the sense originally characterization of tektins from sperm flagellar doublet microtubules. J. Cell Biol. 104, 1069-1075. formulated by Sussman & Newell (1972). LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J'. biol. This work was supported by USPHS grant GM20644 (to Chem. 193, 265-275. R.E.S.) and GM35648 (to R. W. Linck) from the National MERLINO, G. T., CHAMBERLAIN, J. P. & KLEINSMITH, L. J. (1978). Institute of General Medical Sciences. I thank Dr Richard W. Effects of deciliation on tubulin messenger RNA activity in sea Linck, University of Minnesota, for the generous supply of urchin embryos. J. biol. Chem. 253, 7078-7085. tektin antibodies and for many valuable discussions during the NEMER, M. (1986). An altered series of ectodermal gene expressions course of this work, and both him and Dr Jan Norrander for accompanying the reversible suspension of differentiation in the their very constructive comments on the manuscript. zinc-animalized sea urchin embryo. Devi Biol. 114, 214-224. NEMER, M., WILKINSON, D. G. & TRAVAGLINI, E. C. (1985). Primary differentiation and ectoderm-specific gene expression in References the animalized sea urchin embryo. Devi Biol. 109, 418-427. NORRANDER, J. M., STEPHENS, R. E. & LINCK, R. W. (1988). Levels AMOS, W. B., AMOS, L. A. & LINCK, R. W. (1985). Proteins closely of ciliary 55kD tektin mRNA during sea urchin embryo similar to flagellar tektins are detected in cilia but not in development. J. Cell Biol. 107, 20a. cytoplasmic microtubules. Cell Motil. 5, 239-249. O'FARRELL, P. H. (1975). High resolution two-dimensional AMOS, W. B., AMOS, L. A. & LINCK, R. W. (1986). Studies of tektin electrophoresis of proteins. J. biol. Chem. 250, 4007-4021. filaments from flagellar microtubules by immunoelectron RIEDERER-HENDERSON, M. A. & ROSENBAUM, J. L. (1979). Ciliary microscopy. J. Cell Sci. Suppl. 5, 55-68. elongation in blastulae of Arbacia punctiilata induced by trypsin. AUCLAIR, W. & SIEOEL, B. W. (1966). Ciliary regeneration in the sea Devi Biol. 70, 500-509. urchin embryo: Evidence for a pool of ciliary proteins. Science STEFFEN, W. & LINCK, R. W. (1988). Evidence for tektins in 154, 913-915. and axonemal microtubules. Pwc. natn. Acad. Sci. BURNETTE, W. N. (1981). "Western blotting": Electrophoretic U.S.A. 85, 2643-2647. transfer of proteins from sodium dodecyl sulfate-polyacrylamide STEPHENS, R. E. (1972a). Studies on the development of the sea gels to unmodified nitrocellulose and radiographic detection with urchin Stmngylocentrotus droebachiensis. I. Ecology and normal antibody and radioiodinated protein A. Analvt. Biochem. 112, development. Biol. Bull. mar. biol. Lab., Woods Hole 142, 132-144. 195-230. STEPHENS, R. E. (19726). Studies on the development of the sea BURNS, R. G. (1973). Kinetics of regeneration of sea-urchin cilia. J. urchin Strongylocentrotus droebachiensis. III. Embryonic synthesis Cell Sci. 13,55-67. of ciliary proteins. Biol. Bull. mar. biol. Lab., Woods Hole 142, BURNS, R. G. (1979). Kinetics of regeneration of sea urchin cilia. 489-504. II. Regeneration of animalized cilia. J. Cell Sci. 37, 205-215. STEPHENS, R. E. (1977). Differential protein synthesis and utilization FAIRBANKS, G. T., STECK, T. & WALLACH, D. F. H. (1971). during cilia formation in sea urchin embryos. Devi Biol. 61, Electrophoretic analysis of the major peptides of the human 311-329. erythrocyte. Biochemistry 10, 2606-2617. STEPHENS, R. E. (1978). Primary structural differences among GONG, Z. & BRANDHORST, B. (1987). Stimulation of tubulin gene tubulin subunits from flagella, cilia, and the cytoplasm. transcription by deciliation of sea urchin embryos. Molec. cell. Biol. Biochemistry 17, 2882-2891. 7, 4238-4246. STEPHENS, R. E. (1986). Isolation of embryonic cilia and sperm GOROVSKY, M. A., CARLSON, K. & ROSENBAUM, J. L. (1970). flagella. Meth. Cell Biol. 27, 217-227. Simple method for quantitative densitometry of polyacrylamide STEPHENS, R. E. (1987). Enhanced tektin synthesis and ciliary length gels using fast green. Analyt. Biochem. 35, 359-370. in zinc-animalized sea urchin embryos. J. Cell Biol. 105, 95a. HARLOW, P. & NEMER, M. (1987). Coordinate and selective /?- STEPHENS, R. E., OLESZKO-SZUTS, S. & LINCK, R. W. (1989). tubulin gene expression associated with cilium formation in sea Retention of ciliary nine-fold structure after removal of urchin embryos. Genes Dev. 1, 1293-1304. microtubules. J. Cell Sci. 92, 391-402. INOUE, S. (1981). Video image processing greatly enhances contrast, SUSSMAN, M. & NEWELL, P. C. (1972). Quantal Control. In quality, and speed in polarization-based microscopy. J. Cell Biol. Molecular Genetics and Developmental Biology, (ed. M. 89, 346-356. Sussman), pp. 275-302. Englewood Cliffs, New Jersey: Prentice- LAEMMLI, U. K. (1970). Cleavage of structural proteins during the Hall, Inc. assembly of the head of bacteriophage T4. Nature, Land. 227, 680-685. LlNCK, R. W. (1976). Flagellar doublet microtubules: fractionation (Received 20 October 1988 - Accepted S December 1988)

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