Kinetics of the regeneration of sea-urchin cilia : II regeneration of animalized cilia

Cilia of sea-urchin embryos will regenerate following deciliation, even in the presence of inhibitors of protein synthesis (Auclair & Siegel, 1966; Burns, 1973), indicating that there is a significant pool of ciliary precursors. By contrast, similar inhibitor experiments indicate that the complete regeneration of the flagella of Chlamy-domonas (Rosenbaum, Moulder & Ringo, 1969; Coyne & Rosenbaum, 1970; Farrell, 1976), Euglena (Rosenbaum & Child, 1967) and Didymium (Kerr, 1972) is dependent upon protein synthesis. Sea-urchin embryos are therefore particularly amenable for studying cilia regeneration, since the kinetics of growth can be expected to be less complex as protein synthesis is not essential. However, despite the presence of a large precursor pool, the kinetics are not simple, as the cilia grow with deceleratory kinetics to a maximum length, after which further growth ceases even though the precursor pool is not exhausted (Burns, 1973). In addition, sea-urchin blastulae bear an apical tuft of cilia which are 2–3 times longer than the normal cilia, and these will also regenerate to their initial (longer) lengths (Bums, 1973), indicating independent control over the lengths of both the normal and apical cilia. The extent of this apical tuft can be increased by a variety of agents added at fertilization (Lallier, 1964) or on hatching (Riederer-Henderson & Rosenbaum, 1975), to yield animalized embryos. There are therefore within the sea-urchin embryo mechanisms which determine the length of normal cilia, the length of the apical tuft, and which regulate the extent of the apical tuft. The current work concentrates primarily on the kinetics of regeneration of the cilia of the apical tuft, using embryos which had been animalized by treatment with trypsin at the blastula stage. Some of the results have been presented earlier (Burns, 1977).

The methods for fertilizing and culturing sea-urchin embryos, the deciliation procedure, and the preparation of detached cilia as a dried smear on a microscope slide have been described elsewhere (Burns, 1973). This work used Arbacia punctulata embryos which were grown at 20 °C at a dilution of 1:30o (v:v), and which hatched about 12 h after fertilization. The lengths of the dried cilia were measured directly using a micrometer eyepiece (final magnification × 800) rather than from photographic enlargements.

Embryos were animalized using the procedure of Riederer-Henderson & Rosenbaum (1975, 1978). Embryos were treated with trypsin (Sigma Chemical Company: Type II/50 μg ml^-1) from shortly after hatching for an additional 12–20 h. This prolonged treatment with high concentrations of trypsin is necessary to induce extensive animalization (Reiderer-Henderson & Rosenbaum, 1979).

The rate of protein synthesis was determined by incubating extensively washed animalized embryos with L-[4,5-³H(N)]leucine (25μCiml^-1; New England Nuclear: specific activity 60 Ci/mmol). The incubation was stopped by adding an equal volume of 1 M KOH, incubating at 50 °C for 10 min to hydrolyse any charged tRNAs, and adding hydrogen peroxide to a final concentration of 75 mM to decolorize the sample. The protein was then precipitated with trichloroacetic acid (final concentration 10%), and the precipitate filtered onto glass fibre disks (GFA, Whatman 21 cm), washed with 5% trichloroacetic acid and ethanol. The filters were placed in scintillant (Aquasol, New England Nuclear) and counted in a Beckman scintillation counter.

Normal, untreated Arbacia blastulae have cilia with a mean length of 18 am and a small number of longer cilia forming the apical tuft (Fig. 1A). On treating with 50 μg ml^-1 trypsin these embryos become animalized and the proportion of longer cilia increases (Fig. 1 B). Embryo development is arrested by the addition of trypsin : treated blastulae fail to gastrulate, and if the trypsin is added to gastrulae animalization occurs, although the embryos do not form prisms. It is important to note that the distribution of cilia length of the animalized embryos shows a complete gradation between the normal and totally animalized lengths, and is not a simple bimodal distribution.

Untreated blastulae and embryos incubated in 50 μg/ml trypsin for 18 h were deciliated, washed, and transferred to fresh seawater. The regeneration kinetics of both the control and the animalized embryos were examined (Fig. 2 A). In both cases there was a lag phase followed by a period of rapid growth which slowed as the cilia elongated. These kinetics are identical to those found with control and animalized embryos of Triplicates gratilla (Burns, 1973). The kinetics of regeneration of the animalized cilia differed slightly from those of the control cilia, in that they grew longer and the kinetics appeared to be biphasic, with the initial rate slowing to a second, constant rate of elongation. There was considerable variation between different cultures in how sharply the initial phase changed to the second slower phase (compare Figs. 2 A, 4 (control), and 5 (control)), although the biphasic kinetics became more apparent by analysing the length distribution at each point during regeneration. The determination of a mean length assumes that the distribution is homogenous and approximately normal, yet the length distribution of the animalized cilia is not gaussian (Fig. 1 B). The longest 20% and the shortest 20% were arbitrarily considered as quasi-normal distributions, and the increase in mean length of these sub-populations is shown in Fig. 2B. The shortest 20% include all those cilia at the vegetal pole and their regeneration kinetics are identical with those of untreated embryos. By contrast, the longest 20% show distinct biphasic kinetics: an initial linear phase and a greatly reduced second linear phase (Fig. 2B).

These biphasic kinetics suggest that Arbacia embryos may contain a precursor pool which is inadequate for the complete regeneration of cilia to the animalized length. Consequently, the inhibition of protein synthesis should have little effect on the first phase but should abolish the second slower elongation rate. Emetine is an effective inhibitor of protein synthesis (Grollman, 1966) and a dose/response curve indicated that 10 μM emetine inhibited protein synthesis without any detectable injury to the embryo (data not presented). The rate of protein synthesis was inhibited 93% by 10/4M emetine (Fig. 3), which probably represents total inhibition of cytoplasmic protein synthesis as emetine is only partially effective against mitochondrial protein synthesis (Chakrabarti, Dube & Roy, 1972).

The addition of 10 μM emetine to regenerating animalized embryos totally eliminates the second elongation phase (Fig. 4). When emetine is added at 30 min the cilia continue to elongate, although the rate is inhibited by approximately 25%, and then the rate of increase abruptly levels off. The addition of emetine at 70 min, shortly before the commencement of the second phase, results in an immediate inhibition of elongation. The difference between the plateau values resulting from the addition of emetine at 30 min and at 70 min may reflect the rate of protein synthesis occurring during these 40 min. This rate, calculated from the plateau values, is equivalent to o-i6/tm/min of cilia, although the rate of the second phase of the untreated control is only 0.07 μm/min.

The inhibition by emetine of the second phase strongly suggests that the precursor pool is only adequate to sustain the initial phase and that the continued growth of animalized cilia towards their initial length is dependent upon protein synthesis. Consequently, if emetine is added at the time of deciliation, the cilia should regenerate only to the extent of the precursor pool, and if the embryos are then deciliated for a second time there should be no further cilia growth. Indeed, the addition of 10 μM emetine at the time of deciliation results in a 25% inhibition of the initial elongation rate and the complete inhibition of the second phase at a plateau value of 13.5 μm (Fig. 5). The embryos were deciliated a second time, coincident with the initiation of the second phase (arrow, Fig. 5), and then monitored for their ability to regenerate cilia. Despite the prediction that there would be no further growth, the cilia of the emetine-treated and the control embryos both regenerated. The initial phase was at the same rate as the equivalent initial phase during the first regeneration, and in the emetine-treated embryos growth abruptly ceased when the cilia had attained 7 μm, half the plateau value of the first regeneration cycle (Fig. 5). This observation indicates that although the precursor pool appears to be exhausted by the end of the first phase when the embryos are grown in the presence of emetine, this is in fact an oversimplification. Furthermore, the initial phase of the untreated embryos terminates at approximately 20 μm while the plateau value of the emetine-treated embryos is 13.5 μm, indicating that the precursor pool was apparently expanding in the control at a rate equivalent to 0.065 μm/min, while the rate of elongation during the second phase of the untreated embryos was only 0.035 μm/min. A similar discrepancy between the apparent rate of synthesis of the limiting precursor determined from the rate of elongation during the second phase of the control embryos and from the plateau values following treatment with emetine was described earlier (Fig. 4).

The animalization induced by treatment with trypsin differs from all other animal-ization procedures, with the exception of the use of concanavalin A (Riederer-Henderson & Rosenbaum, 1975, 1979) in that it is effective at the blastula stage: it is therefore possible to switch on experimentally the events which lead to animalization. A disadvantage of the procedure is that high concentrations of trypsin for prolonged times are required for complete animalization, as assessed by the extent of the apical tuft of cilia. Therefore although the animalized embryos are morphologically similar to the untreated blastulae, with the exception of the extent of the apical tuft, they cannot be compared directly with the untreated embryos. Despite this limitation, studies of the regeneration of the cilia of animalized embryos may help elucidate the mechanism by which the apical cilia of normal embryos grow to approximately three times the length of the remaining cilia.

Normal cilia regenerate with deceleratory kinetics, so that they approach their maximum length asymptotically (Fig. 2; and Burns, 1973). By contrast, although animalized cilia regenerate at the same initial rate, the kinetics slow to a second, linear rate so that the initial cilia length is only slowly attained. Detailed analysis of the regeneration kinetics indicates that the growth is biphasic, an initial linear rate, equal to the initial elongation rate of normal embryos, being followed by a second, slower linear rate. The regeneration of animalized cilia is therefore significantly different from that of the normal cilia of untreated embryos.

The simplest explanation for the biphasic kinetics is that there is a precursor pool which is exhausted during the initial phase, and that the second phase represents the de novo synthesis of the limiting precursor(s). The addition of emetine, at concentrations which markedly inhibit the incorporation of [³H]leucine, totally inhibits the second phase, while reducing the rate of the initial phase by about 25%. However, when emetine-treated embryos are deciliated a second time, there is a partial regeneration of the cilia although the cilia had stopped growing prior to the second deciHation; the length attained in the presence of emetine does not therefore represent the full extent of the precursor pool. Following the second deciliation, the cilia grow to approximately half the plateau value of the first regeneration, indicating that at least one third of the total precursor pool is not assembled during the first cycle. Clearly the plateau values following successive deciliations would form a geometric series, so that the total pool would equal twice the plateau value of the first cycle. This residual pool is probably necessary to maintain the assemblydisassembly equilibrium in favour of the assembled cilium.

The observation that only half of the precursor pool is utilized does not account for the 2-fold discrepancy in the apparent rate of synthesis of the limiting precursor(s), when measured by the rate of elongation during the second phase compared with the difference in length after inhibiting protein synthesis at different times; e.g. 0.07 μm/min:0.16 μm/min (Fig. 4), and 0.035 μm/min:0.065 μm/min. The reason for this discrepancy is unclear. One possibility is that emetine disturbs the equilibrium between assembly and disassembly so that a smaller residual pool is required, or if the limiting precursor requires to be activated, emetine may stimulate this modification. An alternative possibility is that the size of the residual pool required may be dependent upon the length of the assembled cilium: a proportionately larger pool may be required to maintain a longer cilium. Some evidence supports this alternative as the break between the initial and the second phase occurs at 38.5 μm in the 20% fastest growing cilia while the original mean length of the 20% longest cilia was 35 μm, assuming that the cells with initially the longest cilia give rise on regeneration to the longest (fastest growing) cilia.

One distinctive feature of the emetine-treated cultures is the abrupt transition from the initial phase to the plateau value. This would suggest that all the cells have an identical amount of the limiting component. There is, however, a considerable variation in the length of the cilia of the plateau value (e.g. 13.2 ± 3.45 μm). Furthermore, the cilia at the apical pole of normal embryos grow faster than those at the vegetal pole (M. M. Pratt, personal communication), and in animalized embryos a limited number of in situ measurements indicate that the cilia at the animal pole grow faster than those at the equator. One possibility is that the limiting component not only determines the final cifia length but also the rate of elongation during the initial phase. However, the initial rate of elongation during the second cycle of regeneration in the presence of emetine is the same as during the first (0.116 μm/min: 0.134 μm/min), although the size of the residual pool has been significantly reduced. While this observation demonstrates that the amount of the lengthdetermining component does not determine the initial rate of elongation, the possi-biHty remains that cells whose cilia grow faster, i.e. those towards the apical pole, contain a larger amount of the limiting component. The abrupt transition to the plateau value probably results from such a spatial variation in the amount of the limiting component and the rate at which cilia regenerate, together with the co operative nature of the assembly, such that the pool is effectively depleted twice as fast as the rate of elongation, because of the requirement to maintain a residual pool.

Measuring the pool size by examining the extent of regeneration in the presence of inhibitors of protein synthesis will quantitate only the amount of the limiting component, while the axonemes of cilia and flagella contain at least 150 proteins (Luck, Piperno, Ramanis & Huang, 1977). There is no direct evidence as to the identity of the limiting component in Arbacia embryos, but in Strongylocentrotus droebachiensis, Stephens has shown that an axonemal protein associated with the ribbon between the A- and B-microtubule of the outer doublet is preferentially synthesized during regeneration (Stephens, 1977). When regenerating embryos were incubated with pCjleucine, this component (component-20) was the predominant labelled protein, although it represents less than 2% of the total axonemal protein, and on regeneration for a second time, but in the absence of label, the specific activity of component-20 fell by 85% (Stephens, 1976, 1977). One other component, component-io, which is probably the nexin linkages between adjacent outer doublets, was also preferentially synthesized, but not to the same extent as component-20. Stephens suggests that the quantal synthesis of component-20 could limit the length of the protofilament ribbon and consequently limit the length of the axoneme.

It is probable that the pool of component-20 is also limiting in Arbacia punctulata embryos, and that regeneration in the presence of emetine reflects the size of this pool. There is, however, a significant difference between A. punctulata and S. droe-bachiensis: Stephens observes that the cilia of normal embryos will regenerate to only one third of their initial length in the presence of puromycin (personal communication), while normal Arbacia embryos will completely regenerate in the presence of emetine (unpublished observation) and animalized embryos will partially regenerate. These results suggest that the size of the pool of the limiting component may vary between sea-urchin species.

The regulation of the regeneration of the animalized cilia may reflect that of the apical cilia of normal embryos, with the reservation already discussed that the animalized embryos cannot be considered identical to the untreated embryo because of the time and concentration of trypsin necessary to induce animalization. It has been suggested that the kinetics of the regeneration of normal cilia are determined by the rate of transport of precursors to the distal tip plus an additional length-determining mechanism (Bums, 1973). Child has recently proposed an alternative mechanism in which some of the ciliary structure is assembled at the distal tip and the remainder at the proximal end (Child, 1979), which would necessitate a continuous reorganization of the assembled structure during regeneration. He also proposes that the distal- and proximal-assembled elements are connected by radial or circumferential linkages which continually make-and-break, and that the final length is determined by the lability of these linkages, so affecting the sliding of the distal- and proximal-assembled elements.

The initial phase of the regeneration of the animalized embryos is approximately linear and at the same rate as the initial elongation of normal cilia. This suggests that in the animalized cilia the restriction which results in the deceleratory kinetics of the normal cilia has been eliminated, which in the Child model would be the radial or circumferential linkages. In other words, the length-determining mechanism of normal cilia has been abolished in the animalized embryos. The animalized cilia should therefore continue to grow indefinitely, yet even after 40 h growth the cilia never exceed about 55 μm (data not shown). The mean residual pool of animalized embryos is about 20– 25 μm (Figs. 4> 5) and even in the 20% fastest growing cilia the residual pool is only about 40 μm (Fig. 2B). It is proposed that in the animalized embryos the length-determining mechanism of normal embryos has been abolished and has been replaced by the ability of the cells to synthesize the limiting component, possibly component-20. The observation that the cilia never exceed about 55 μm may indicate that the residual pool continues to expand until it reaches a critical concentration when further synthesis of the limiting component is switched off. In normal embryos there is presumably a high concentration of the limiting component in the cells at the apical tuft and in these cells the length-determining mechanism is inoperative. Two processes must occur during animalization: the abolition of the length-determining mechanism and the continued expansion of the pool of the limiting component. As the latter occurs at a slow rate (0.065–0.16μm /min), the prolonged animalization treatment may represent in part the time required to synthesize more of the limiting component.

I should like to thank the Medical Research Council and the Steps-Towards-Independence Program of the Marine Biological Laboratory for supporting this work, and to thank colleagues at the Marine Biological Laboratory for their interest and encouragement.