a discussion on cytoplasmic organelles || the flagellar apparatus as a model organelle for the study...

The Flagellar Apparatus as a Model Organelle for the Study of Growth and MorphopoiesisAuthor(s): John RandallSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 173, No.1030, A Discussion on Cytoplasmic Organelles (Apr. 15, 1969), pp. 31-55Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/75739 .

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Proc. Roy. Soc. B. 173, 31-62 (1969)

Printed in Great Britain

The flagellar apparatus as a model organelle for the study of growth and morphopoiesis

BY SIR JOHN RANDALL, F.R.S.

Department of Biophysics, King's College, London

[Plates 8 to 11]

With an Appendix Temperature control apparatus used in flagellar

regeneration experiments

BY H. R. MUNDEN AND P. H. PREST

Medical Research Council, Biophysics Research Unit, Department of Biophysics, King's College, London

1. INTRODUCTION

During the last five years a small group in this Laboratory has been studying the potentialities of the flagellar apparatus of Chlamydomonas reinhardii for the study of growth and morphopoiesis. The results have been recorded in a number of papers (Randall et al. I964; Warr et al. I966; Hookes, Randall & Hopkins I967; Randall et al. I967). It is self-evident that the understanding of both growth and morphopoiesis in physical chemical and biological terms will most readily be brought about by the examination of the least complex biological systems, i.e. those that contain the fewest chemical and structural entities and are under the control of a strictly limited number of genes; not for preference a multicellular organism or organ, or even a single cell; but rather a virus or organelle. One would hope by this means to limit the number of genes implicated to something less than 102. An outstanding analysis of the morphopoiesis of T-even bacteriophages has been carried out in recent years by workers in Geneva and Pasadena (see, for example, Kellenberger I964; Kellenberger & Boy de la Tour I965; Epstein et al. I963;

Edgar & Wood I966). In the present investigations the methods and techniques of genetics, of optical and electron microscopy (in conjunction with optical diffraction studies of electron micrographs), radioautography and, to some extent, biochemistry have been used.

During the last 15 years the structure of cilia and flagella, their basal bodies and associated cortical structures in various organisms (see, for example Fawcett I96I;

Gibbons & Grimstone I96o; Satir I965; Ringo I966, I967; Cavalier-Smith I967;

Allen I967) have been investigated in some detail. Figure 20, plate 8, illustrates the appearance of the living organism in motion and figure 21 the fine structure of the flagellar apparatus (PA) of the alga Chlamydomonas reinhardii, a biflagellate

[ 31 ]



32 Sir John Randall (Discussion Meeting)

member of the Chlorophyceae. The FA consists essentially of two normally motile flagella F, external to the cell (figure 20) and two internal basal bodies (BB). Each basal body is joined to its flagellum by a transition region TR (figure 21). The basal bodies (BB) lie in the main diametral plane of the organism and are joined by a fibrous band (FB) (figure 21). The external flagellum, bounded by a membrane continuous with the plasma membrane, contains the axoneme and its subsidiary structures. We shall be concerned chiefly with the basic features of the axoneme of the flagellum, i.e. the two central fibrils and the nine outer pairs. Each of these fibrils is apparently tubular in form and generally similar to the microtubules found in many types of cell (e.g. Tilney & Porter I965; Tilney & Porter I967; Peters & Vaughin I967; Hepler & Newcomb I964). The appearance in the electron microscope of an intact fragment of tubule (outer pair) from a disrupted negatively stained flagellum of C. reinhardii is shown in figure 24a, plate 9. The structural interpretation of micrographs of flagellar tubules has been discussed by Grimstone & Klug (I966) and Hookes, Randall & Hopkins (I967) and will be referred to subsequently. For fuller details of the fine structure of the FA of C. reinhardii, as a whole, reference should be made to Ringo (I966, I967) and to Cavalier-Smith (I967).

Reasons for the choice of C. reinhardii as a suitable organism for the investigation of the morphopoiesis and growth of the FA have been given in our earlier papers. Notable among these are the distinctness of the mature organelle structure in the electron microscope; the existence of a sexual (as well as a vegetative) life-cycle which has already been the subject of much genetical study (e.g. Levine & Ebersold I958, I960; Ebersold et al. I962; Sager I955, I965). This has enabled us on the one hand to obtain a substantial amount of new information on the natural history of the FA, both in development and regression (Cavalier-Smith I967); and on the other to use vegetative cells for mutagenesis and haploid gametes for genetic analysis. A distinct disadvantage of the FA for morphopoietic studies is its comparative complexity, which is greater than that of the phages already referred to. It should also be noted that the finer details of flagellar structure (subsidiary fibrils, side-arms, etc.) are not in general regularly reproducible in electron micrographs. Satisfactory comparisons of fine structure of such components in different genetic strains are therefore likely to be difficult.

Our study of growth is more strictly one of regeneration of the external flagellum, i.e. a clearly measurable part of the FA. C. reinhardii shares with a number of other organisms (e.g. Ochromonas, Dubnau I96I; Tetrahymena, Child I965; Ochro- monfas, Astasia and Euglena, Rosenbaum & Child I964 and I967; Peranema, Tamm I966) the property of regenerating new external flagella after the originals have been removed by mechanical agitation, mild chemical treatment or temperature shock. Lewin (I953) appears to have been the first to study regeneration in Chlatmydomonas flagella.

An essential part of the knowledge of organelle morphopoiesis and growth lies in the identification and characterization of its biochemical components,



Flagellar apparatus as a model organelle 33

particularly the proteins. Progress in this direction is largely dependent on the isolation of the whole organelle and its distinctive parts in pure form. No satisfactory preparations of the whole FA have yet been made. Substantial progress has, however, been made in the study of preparations of the external cilia of Tetra- hymena by Gibbons (I963, i965 a, b) and Watson and others (Watson, Alexander & Silvester I964; Watson & Hynes I966). Studies of the flagella of C. reinhardii are now being made by Dr M. Jacobs who will describe later in this Discussion his investigations of the axoneme proteins of normal flagella and of a (9 + 0) mutant

(pfl9b) (p. 61).

II. THE NATURAL HISTORY OF THE FLAGELLAR ORGANELLE IN

CHLAMYDOMO0NAS REINHARDII

This history has been examined by Cavalier-Smith (I967). While incomplete in a number of respects the following results appear to be established and are taken mostly from Cavalier-Smith (I967) and Randall et al. (I967):

(i) The flagellar apparatus (PA) appears to be assembled sequentially from basal bodies (BB), transition region structures (TR) and external flagellum in that order (figure 22).

(ii) The earliest stage in basal body assembly for which there is clear evidence is a cylinder consisting of nine singlet microtubules surrounding a central 'cartwheel' structure; the nine single :microtubules probably correspond with the A subfibres of the mature PA. Many basal bodies at this stage are shorter than when mature.

(iii) B and C subfibres are added to the above structure to form complete triplets which grow to a length of 0 4 Vm, thus forming the mature basal body.

(iv) The A and B subfibres of the basal body subsequently extend to form the outer nine doublets of the external flagellum. The transitional region (TR) of the flagellum (figures 21 and 22) is formed before the (9+2) axoneme proper.

(v) In vegetative cells about to divide and in early 4-flagellate zygotes, light microscopy shows that the flagella regress gradually and are not simply broken off. This process appears to take about 30 min. This period of regression if reproducible is less than the corresponding period of regeneration at the same temperature by perhaps a factor of between 5 and 10.

(vi) There is a difference in the mechanism of regression (or its control) in the vegetative and sexual cycles, since basal bodies persist in the vegetative cells, but not in the zygotes, after flagellar regression.

(vii) Results from the sexual cycle where basal bodies disappear in the zygote show that no morphologically identifiable basal body is necessary for formiation of new basal bodies.

(viii) On the other hand, in the vegetative cycle the basal bodies of the daughter cell are formed in the presence of the parental organelles; this does not in itself prove their interdependence. (But see, for example, Robbins, Jentzsch & Micali I968).

3 Vol. 173. B.




(ix) Basal bodies are formed before the roots or striated connexions between the basal bodies of a pair.

(x) During germination of zygotes roots are probably formed before external flagella.

CENTRAL PAIR

OUTER NINE PAIRS

PLASMA MEMBRANE

(F)(

J5TRANSITION REGION [ \

CQ"/~~~~~~~~~~~~~~~~(i

BASAL BODY

TRIPLETS ABC (i)

ca RTWHEE L (cwY)

FIGURE 22. Diagrammatic representation of known features of FA of C. reinhardii. Left-hand diagram: detail. Right-hand diagram: schematic representation of three main features: basal body, BB; transition region, TR; and flagellum, F. (i) Tubules ABC of basal body; (ii) tubules AB of basal body and outer nine pairs. C W, cartwheel structure of basal body. (Based on Cavalier-Smith I967.)

In more general terms: (xi) The FA is a cortical polar structure, particularly the basal body (BB) and

transition region (TR) from which the flagellum always grows in an outward direction. For comparison note that the microtubules of cell spindles diverge from centrioles which are structurally closely homologous with basal bodies, but do not possess structures analogous to those of the transition region of the FA. The interesting question as to whether bacterial flagella also spring from basal bodies has recently been re-investigated by Van Iterson, Hoeniger & Van Zanten (I966).

However, the formation of numbers of free or unorganized microtubules within




many types of cells (already referred to) appears to take place in the absence of any such complex. It is a reasonable hypothesis that the cortical regions of the FA (BB, TR and the niearby plasma membrane) contains substanlces controlling its overall organization. Such molecules are not necessarily structurally identifiable as part of the FA, since a very thin layer might suffice from a functionial standpoint. Adsorption to the main structure(s) could be a condition of allosteric control.

III. THE GENETIC CONTROL OF FLAGELLAR STRUCTURE IN

CLILAMYDOMONAS REINHARDII

One of our first objectives was to search for mutant strains with structural abnormalities in the flagellar apparatus, and thus to gain some insight into the genetic control of structure. Previous studies of non-motile strains of the related species C. moewusii (Gibbs, Lewin & Philpott I958) had failed to reveal such abnormalities. In our first experiments a non-motile mutant of C. reinhardii was isolated and found to possess a clearly observable structural abnormality. The central pair of fibrils of the (9 + 2) axoneme assembly was replaced by an irregular core of apparently disorganized material (Randall et al. I964). Subsequently various other mutants have been isolated and analysed genetically (Warr et al. I966), several of which together with others isolated by R. A. Lewin have been shown to have a similar defect. In all over eighty mutations affecting the flagella of C. reinhardii have been isolated in our laboratory and elsewhere. The method of isolation is such that only non-motile mutants or mutants with much impaired motility are likely to be detected. They are thus all recognizable in the light microscope as abnormal, but detailed genetic and fine structural analysis, is required to distinguish and characterize the mutations satisfactorily. Table 1 classifies the mutants.

TABLE 1. FLAGELLAR MUTANTS OF CHLAMYDOMONAS REINHARDII

number number mutant class of strains of loci

central pair mutants (non-leaky) 1.5 3 central pair mutants (leaky) 6 1 swollen flagellum mutants 5 ulnknown flagella-less mutants 8 unknown stumpy mutants 6 unknown short flagellum mutants 11 unknown long flagellum mutants 2 2 impaired motility mutants (no structural 29 10 or more

abnormality) mutants with abnormal numbers of flagella 2 2 suppressor mutant 1 1

Reference should be made to Warr et al. (I966) and Randall et al. (I967) for a fuller account of the genetic studies. The mutants with abnormal numbers of flagella have recently been described more fully by Warr (1 968). The following facts are relevant to the present Discussion.

3-2




Centre pair mutants

Twenty-one mutants are so far known to have disrupted central tubules. In 14 of these, all of which exhibit the visual properties of paralysed straight flagella, electron microscopy has shown that both the central tubules had become disorganized and were replaced by a single irregular column of moderately densely staining material. For convenience we refer to this appearance as the (9+0) pattern to distinguish it from the (9 + 2) arrangement of tubules in normal flagella. Six of these mutations had oiiginally been isolated by Lewin and had been mapped genetically by Ebersold and his colleagues (Ebersold et al. I962). This has provided us with a means of mapping other centre pair mutants. Eleven strains of paralysed flagella in all were analysed genetically by Ebersold et at. and found to fall on 10 different linkage groups. Seven of these mutants appear to be structurally normal. However, examination of eight of the new strains isolated by us (and structurally designated as (9 + 0) mutants) indicates that each of these new mutations is either at or closely linked to a known locus (III, II, X and IV). Moreover, each of these eight (9 + 0) mutations was found to involve mutation at a single gene. Detailed investigation of the 21 central pair mutants of table 1 shows that they are of two kinds: those (15 in all) with the (9+0) structure already described; and another group of six leaky mutants which can be detected either by the light microscope or the electron microscope. In a leaky strain a small proportion of the cells either rotate without forward movement or alternatively rotate with forward movement. In some cases a still smaller proportion of fully motile cells may be present. It is evident that both these types of movement result from functional dissymmetry of the two flagella. Demonstration of the degree of dissymmetry in terms of fine structure between the two flagella of a single organism is technically difficult. In general, however, the electron microscope has revealed in the leaky strains flagella with normal structure (9+2); flagella with one central tubule normal and the other abnormal (9+1) arrangement; and also flagella with both central tubules disorganized (i.e. (9 + 0) arrangement). Examination of various strains shows that where disorganization exists, it probably extends over the whole length of the flagellum. pf 20 and pf20A appear to be the most leaky mutations.

Suppressor of tubule mutations

As stated in Warr et al. (I966), interactions between genes are likely to represent a basic feature of organelle morphogenesis, and a search was therefore made for suppressors of the centre pair mutations in the form of motile revertants. The results have been described (Warr et at. I966) and it is only necessary to say here that the flagella of the revertant strain show three distinct kinds of structure similar to those that occur in cultures of the very leaky mutants (pf 20 and pf 20 A) viz. (9+0), (9 + 1) and (9+2) arrangements of the axoneme. Genetic analysis suggested that this reversion was due to interaction between the pf 19b allele and an unlinked suppressor (SU 1). Analysis has also been made of the extent to which



Randall Proc. Roy. Soc. B, volume 173, plate 8

FURE 23 ;. (a o() htmcorpso h wle lglao FWmu ta t 'pn1;arado

i- s)

~~TR -

2^; ~BB_

FIGURE 20. Photomicrograph of living C7hlamydomona8 reinhardii in motion. Phase contrast. ( X 2250.)

FIGURE 21. Electron micrograph of flagellar apparatus (FA) of C. reiXnha-rdii/. Longitudinal section. BB, basal body; TR, transition region; FB, striated fibrous band; F, proximal portion of flagellum; N, nucleus, C; chloroplast. ( x 28240.)

selection of examples. Phase contrast. (x 1700.) (Facing P. 36)




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.s S,,.,.,, .,,,. .......... . . . .. ; .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.. .. ...

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%t:,4! : ,. i . , .. . .'. . . . . . :. .# . .

.i, , I<$-tt. ..,.

. . .... . .. ... ..... .

L?;L8

FIGURE 24. (a) Electron micrograph of negatively stained fragment of outer pair of tubules. ( x 220 000.) (6) Optical transform of (a) indicating that the size of subunit in the structure of (a) is 45A.




a b

FIGURE 25. Photomicrographs of regenerating flagella of C. reinhardii after partial deflagellation. (a) to (f): the course of events after the removal of one flagellum. Note the complete regression of the remaining flagellum before regeneration of both begins. (g) to (i): two flagella are shortened by equal amounts-regeneration proceeds at the same rate in both and without any initial regression. ( x 2000.)



Randall Proc. Roy. Soc. B, voZxqne 173, plate 11

., E .. . fl .....

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AF . ......................... ,, Z }; tt'v; . f

E '4f j 4

=' -'< e_ _:.

_l _ _ --'1

_S _!_ _L'li;Bv jl X Jlt [ _ ''1 Z..k

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!B11 S . *':.s _ ' : a t /e t?t .

-:tt3lX ' s _ *f;,..} a T _P_

a _S l_

Z FIGURE 32. (a) to (d): electron micrographs (longitudinal sections) of early flagellar regenera-

tion at t = O, 5, 10 and 15 min approximately in a. reinhardii (wild-type), providing evidence that there is no true lag period. ( x 61000.)




other centre-pair mutants were suppressed and some variation in this respect has been found between different centre-pair loci.

Mutants with swollen flagella Five mutants with swollen flagella have been isolated. None of these has been

examined in detail, but more is known of 'Spon 1' than the other four. Figure 23 (a) to (f), plate 8, shows that a flagellum may grow to full length before swelling begins. Moreover, swelling may start at any point along the length of a flagellum and at different times on each of the pair. Swelling gradually increases to the point where the flagellum is indistinguishable from a sphere. Swollen flagella may break off and are to be found floating in the medium. Each flagellum contains an axoneme that is normal in cross-section. No evidence of axoneme folding has been observed in electron nicrographs, but the relationship of axoneme length to the degree of swelling is not yet clear.

Flacgella-less, stumpy and short flagellum mutants Miss MeVittie has isolated a number of these and has examined the fine structure

of several in the electron microscope (this Discussion, p. 59). A number of these mutants show structural abnormalities in the transition region TR of the FA.

Mutants with long flagella These are of considerable interest but we have no further data to add (Randall

et al. I967).

Mutants with paralysed flagella of apparently normal structure Twenty-nine mutants with either wholly or partially paralysed flagella of

normal length have been isolated by ourselves and others. Although 23 of these strains have been examined in the electron microscope no structural abnormalities of the FA have so far been observed. However, it is important to note that the finer details of flagellar structure are by nlo means easy to demonstrate and it is quite possible that improvements in technique will alter this rather unpromising situation.

Mutants with abnormal number of flagella These have been observed and in one of these the character is evidently inherited

as a single gene mutation (Warr I968). The mutants of this class show defects of cytokinesis; the cells are multinucleate and there is no reason at present to believe that the flagella per se are abnormal.

IV. MACROMOLECULAR BASIS OF FLAGELLAR STRUCTURE

From the work of Cavalier-Smith (I967) it has been shown that the flagellar apparatus FA is assembled sequentially. It therefore follows that morphopoiesis of the organelle takes place as a result of the provision of the appropriate species of




macromolecules at the appropriate sites and at the appropriate times. This is in accord with previous hypothetical considerations. (For discussion of possible assembly processes see Hookes et al. I967.) It is natural to ask in what form are these several macromolecular species: (i) before they are assembled, and particularly (ii) after they are assembled into the structure. (i) is particularly difficult to answer, but it is possible to say something about (ii) because the well-defined mature organelle provides a firm basis for investigation by electron microscope of negatively stained preparations of recognizable structures. The work of Pease (I963) on the tips of rat sperm tails suggested that the basic molecular unit of flagella might be quasi-globular. In 1964 I suggested a unit of about 45 A in size, pointed out the axial arrays of units in the flagellar microtubules of Tetrahymena and proposed a preliminary model with 12 such columnar arrays per microtubule. Meanwhile Klug and collaborators (Klug & Berger I964; Klug & Finch I965, and many succeeding papers) had applied an optical diffraction technique to the interpretation of electron micrographs of negatively stained structures, particularly viruses. In 1966 Grimstone & Klug published results on the flagella of Tricho- nympha and discussed the data, but did not propose a model. In 1967 Hookes et al. employed a computer approach to the problem of microtubule structure and some valuable features emerged which will not be discussed here. Work is being carried out on the tubule structure of Chlamydomonas by Mr D. Chasey, particularly on the application of diffraction techniques (including the convolution camera (Elliott, Lowy & Squire I968)) to the study of the micrographs. No firm detailed conclusions are yet possible, largely because of the known difficulties of interpretation in relation to: (a) images of 3-dimensional objects, (b) distortion of the object, and (c) the variation in the degree of focus and also the degree and distribution of staining that can arise. Figure 24a, plate 9, shows an electron micrograph of part of a flagellar doublet from C. reinhardii and figure 24b its optical transform. On present evidence it is reasonable to propose that much of the flagellar apparatus is composed of quasiglobular protein units, those of the axoneme being some 45 A in size. The meaning of the 180 A layer line in figure 24 is not yet clear. The subunits of the side-arms are also globular in shape. From the complexity of the WA structure as a whole, and from such biochemical results as are available, it is inferred that several different kinds of unit are involved, but how many it is impossible to assess with accuracy. For example, reference to the full fine-structural data, much of which is implicit in figures 21, plate 8, and 22, would enable a count of separately distinguishable elements to be made. But without corresponding biochemical results, including those on enzymes possibly required to form and to link the various subunits and structures together, the required assessment cannot be achieved; we are, all too evidently, some distance from this objective. As will be seen later, it is possible from a morphological standpoint to make a very tenta- tive reconstruction of events in the morphopoiesis of the FA, but in terms of genetic control the information available is still very inadequate indeed.




V. FLAGELLAR REGENERATION IN C. REINHARDII:

PROCEDURES AND RESULTS

The morphopoiesis of the complete FA of C. reinhardii as one continuous series of events occurs in the sexual cycle only. It may now reasonably be inferred that this involves a sequential series of molecular processes carried out at distinctive rates. However, one part only of the over-all process is at present accessible for physicochemical measurement: the growth of the external flagellum. It is technically difficult to make such measurements in the course of either of the distinctive types of life-cycle of C. reinhardii. Consequently resort has been made to the regeneration processes affecting the external flagella after deflagellation (Chen I950; Lewin I953; Hagen-Seyfferth I959; Dubnau I96I; Child I965; Tazmm I966, I967; Rosenbaum & Child I967; Randall et al. I967). Deflagellation of virtually a whole culture is easy, but the study of regeneration of flagella (and by implication its control) is a good deal more complex than might appear. Two apparently different approaches are possible:

(a) Partial deftagellation Partial deflagellation of individual cells by the use of micromanipulative

techniques (or of populations by cruder mechanical means) permits the comparative study of the regeneration of the two (in general, unequal) flagella of one organism. This procedure is potentially valuable for the examination of the degree of independent control exercised by one flagellum in close proximity to its neighbour, but has not yet been fully explored, here or elsewhere. For this reason and because it is referred to in the Discussion, brief reference and illustration only will be made to this aspect which it is convenient to include at this point. Figure 25 (a) to (f), plate 10, illustrates the course of regeneration after the removal of one flagellum. First, the lonlg flagellum regresses completely. Both flagella then regenerate at equal rates to full length. This result is consistent with the obser- vation of Ringo & Rosenbaum (I967) on selected cells from which one flagellum has been removed in toto and the other is of normal length. In addition we have noted (figure 25(g), (h) and (i)) that, when the two flagella are shortened equally, no regression is observed and both grow away at equal rates to complete regeneration. Regeneration of unequal flagella is more complex than that of equal flagella and involves an initial regression of the longer partner.

(b) Complete defiagellation Complete deflagellation of a defined cell population allows the average kinetics

of flagellar regeneration in random samples to be examined. The bulk of our work has been carried out using this method (Randall et al. I967).

Cultural procedures

In broad terms cell samples may be taken from two types of culture: either the rapidly growing so-called logarithmic culture (method L) or the synchronously




dividing culture (method S). There are at least two variants of method L. Rosenbaum & Child (I967) (see also Dubnau I96I) have taken samples from 4-day cultures of various organisms and transferred them to media lacking an essential growth component, biotin. After the expiry of 3 days in the new medium it was found that very little cell division was taking place. In our earlier experiments use was made of the fact that a 3-day culture of C. reinhardii in normal growth medium (NGM) transferred to nitrogen-free medium (NFM) for 17 h did not divide. We have used this method in the current work and have also examined regeneration in NFM after repeated deflagellation.

In addition, we have attempted to achieve cell synchronly using a technique similar to that of Bernstein (I960, I96I). A sample of cells already in rapid growth in NGM is subjected to three alternations of dark and of light (intensity 500 ft. candles) each of 12 h duration and in that order. About 1 h before the end of the final light period the cells are transferred to NFM and kept for 17 h before deflagellation.

Measurement of flagellum length

This has presented some problems in that by the application of the usual optical projection and map-measuring devices it is possible to measure only that part of the length external to the cavities in the cell body from which the two flagella emerge. Knowledge of growth within the cavity and of the depth of the cavity has necessarily had to be obtained from electron microscope measurements. Reference to figure 26 shows that the straight proximal portions of the two flagella may be extended internally to meet at 0, a point thus readily determined from photomicrographs. All measurements of length are now made from 0 and a correction applied for the length OT at time t = 0. T represents the extremity of the rounded

stump of the flagellum which forms immediately after deflagellation and which is

C C

T I

%~~~~~~~

FIGURE 26. Diagram used to explain how flagellar lengths are determnined.



Flayellar apparatus as a model organelle 41

seen in electron microscope preparations fixed within one minute of deflagellation. Measurements from electron micrographs of true longitudinal sections of the FA show that in this embedded material OT = 0 75 yim. This value is, however, sub- stantially shorter than in -the living state on account of shrinkage. From careful measurements on living and on fixed, embedded and sectioned organisms and flagella the value of the shrinkage factor S has been estimated to be 2 18. Con- sequently, to obtain the 'best' values of flagellar lengths in regeneration it is necessary to subtract 0-75S from the observed value. 0-75S is thus an estimate of the length OT in the living organism at time t 0. S, however, is estimated from ' overlap' determinations of flagellar length in the light and electron microscopes. In other words, the accuracy of S depends on the assumption that the basal body and transition region when embedded shrink by the same factor as the external flagellum. This may not be true. The light microscope can deal only with lengths external to each cavity C (figure 26). The study of initial stages of growth and of a possible lag period (see later) demands some such procedure as we have described.

The distribution of flagellar lengths about a mean Whatever method of studying the kinetics of flagellar regeneration is adopted

must depend on values of mnean length in statistically significant samples obtained for different values of time t. Some of our recent experiments have been carried out on 100 organisms per sample, but this number has now been reduced to 75 to restrict the labour involved. Even so a single regeneration curve involves precise measurements on some 3000 to 4000 flagella. The work has been aided by the use of an electromechanical Conti calculating machine and by the use of the Elliott 803 computer.

As implied earlier, work has begun on the assessment of the effect of culture method of flagellar length distribution. It had been assumed in the early stages that the flagellar lengths in a well-grown population (method L above) would be distributed about a mean according to the familiar normal error curve. And, in contrast, that use of 'synchronous' cultures (method S) would produce a selected population in which the lengths would be distributed within narrower, perhaps much narrower, limits. The facts so far available do not agree with these assump- tions.

In figures 27 (a) and (b) normal curves have been fitted to the experimentally observed distributions of flagellar lengths of a culture prepared according to method L. The distribution is abnormal and seemingly leptokurtic, with about 85 % of the observations falling within the limits of + 1 s (? F9 tm). For a normal distribution the corresponding percentage is - 68.

In contrast, our attempt by method S to produce a cell population of flagellar lengths within narrower limits has resulted so far (figures 28(a) and (b)) in a distribution much more nearly normal. The data demonstrate that:

(i) The distribution curve of flagellar lengths is, as is to be expected, a function of the method of culture.




75 -

(a)

bOD

CO 5 10 15 205 10 15 20

mean length (am)

FIGuRE 27. Distribution of flagellar lengths about a mean fitted to normal curve. 'Loga- rithmic' culture (25.5 ?C). (a) Undeflagellated control. (b) Regenerating sample at 120 mi.

(a)

40-

20-

Ft -~-F

(b)

40-

20-

0 10o 20

mean length ([km)

FIGURE 28. Distribution of flagellar lengths about a mean fitted to normal curve. 'Syn- chronous' culture (20 'C). (a) Undeflagellated control. (tL, Lm 11.3.) (b) Regenerating sample at 120 min (t Lm, 10.45.)



Flagellar apparatus a8 a model organelle 43

(ii) The distribution parameters imposed before deflagellation appear to persist during the regeneration process in NFM, in which cells do not divide.

(iii) Method S is so far unsuccessful in its intended purpose. While it is possible that methods of culture, and therefore in effect of cell

selection, do not have an important influence on the fundamental aspects of regeneration kinetics, this cannot be asserted without proper evidence. It may be observed that both types of method (L and S) imply that uniformity in properties of cell division confers uniformity on other cell properties, viz. those of the flagella. Quite apart from the fact that cell division must affect the flagellum abruptly, this is an assumption which may not be true. Many basic experiments remain to be done to define the cell, and therefore the organelle, population ab initio. It should then be possible to investigate the influence of the population parameters on the regenerating organelle system.

Our results are therefore dependent on the suitability or otherwise of the culture and sampling methods so far adopted.

'

0 Ci

o O0c 13.5 C

-0-4 - 39?C 320C

I II I I I I I I I1 0 40 80 120 160 200

time (min)

FIGUnRE 29. Semi-log plot of flagellar regeneration in C. reinhardii showing the approximately linear relationship between log (Lm -L) and t where L is the maximum length. Note the effect of temperature on the regeneration rate. (From Randall et al. I967.)

Regeneration kinetics after complete deflagellation Figure 29 taken from Randall et al. (I967) typifies a number of features which it

is useful to note before considering the more accurate and comprehensive data of recent experiments:

(a) There appeared to be an initial lag period of 10 to 20 min according to conditions. This is now known to be wrong.




(b) Regeneration proceeds such that dL/dt decreases with time and eventually

approaches zero, thus giving rise to the conception of a 'final' or maximum length,

Lm. (c) The results obeyed approximately under the conditions described the

empirical relation (Lm-L) Lm e-kt, (1)

or loglo(Lm-L) 0.4343kt+C (2)

where L represents the length at time t. This relation was shown to be in accord

with first-order reaction kinetics.

/ _ ~d e f

t~~~~~~~

T-

FIGU:RE 30. (a) to (i): possible types of regeneration curve (diagrammatic).

(d) Over the range of 13 to 39 0C regeneration proceeded faster the higher the

temperature. From data embodied in figure 29 a rough value of 'activation energy'

was derived, possibly related to the rate-limiting reaction of a complex process

(value 575 x 103 cal mole- deg-'). We are now extending this work in directions already indicated. Since the

experiments are still in progress complete analysis is not possible and a selection

only of results will be given. First consider diagrammatically some of the possibilities to which the regenera-

tion kinetics might conform. In figure 30(a) to (i) four features are selected for

emphasis. (1) In (a), (d) and (g) regeneration begins immediately and rises sharply

from the origin (i.e. no lag period). (2) (b), (e) and (h) depict a time lag period. (3) In (c), (f) and (i) there is no time lag, but the rate of regeneration at first

increases to a steady value. This is followed in all cases (a) to (i) by a sharp decrease

in rate. (4) The remaining question posed in figure 30 is not so much whether the

rate of regeneration settles down to a steady value, but whether it is positive as in



Flaqellar apparatus as a model organelle 45

(a), (b) and (c); zero, as in (d), (e) and (f) or negative as in (g), (h) and (i). In our preliminary experiments the regeneration curves seemed to conform most with (e). At that time electron microscope studies of the early stages had not been carried out. The latest experiments indicate that results conform most closely to the group (a), (d) and (g). It -is not yet possible to define the conditions for which (dL/dt)t, large is positive, zero or negative. Curves similar to (a) and (g) have been observed and there is no reason to doubt that intermediate stages could be found.

0

F~~ ~~~ / *> 0 0*0

0 0

e /

0 50 100 150 200 _ 1001% 2000 3000

time (min)

FIGURE 31. Comparison of regeneration in (o) C. reinhardii wild-type (32 'C) and (*) mutant pfl9b at 25-5 'C.

Moreover, it has been observed that the mean lengths of flagella in sample populations of 'normal' organisms (i.e. undeflagellated) in NFM change slowly with time. In one instance the mean length declined by 0-4 m in 24 h. The slow change of length with time is thus found in normal samples as well as deflagellated ones.

Figure 31 compares regeneration curves at 25-5 'C for wild-type and for the (9+0) mutant pfl9b, the flagella of which are seen to grow more slowly to approximately the length of the wild-type. These curves summarize light microscope data only and no deductions can be made from them about the lag period.

Lag period As explained previously, the existence or otherwise of a true lag period can only

be decided from an electron microscope study of appropriate accurate longitudinal sections. This has now been done by Mr J. M. Hopkins. The results botll in the form of micrographs (figure 32 (a) to (d), plate 11) and as a plot of length against time (figure 33) are unequivocal. Growth starts immediately from the stump that remains after deflagellation, and appears to be approximately linear with time over this small range of values.




The complete regeneration curve and the fitting of data

As a result of overlapping regeneration measurements on the same sample in both the EM and LM it has been possible, by use of the shrinkage factor S already discussed, to normalize the two sets of data and thus to construct the complete

regeneration curve from t- 0 to t 3000 min in a number of instances. An example of such a curve is given in figure 34. This and other recently obtained data have been subjected to least squares analysis and the curves drawn through the points represent the nearest approach to a 'best fit' that we have obtained. For this purpose it has of course been necessary to assume an empirical expression for the growth curve.

7.5-

5-0 -~~~~~~~~~~~~

bO

e0

2 25

() ~~~~~20 40

time (min)

FiG~uRE 33. Plot of data derived from man-y micrographs similar to those of figure 32.

Equations (1) and (2) used on the earlier data (e.g. figure 29) do not wholly account for the real but slight changes of length now known to occur in the later

stages of regeneration and to which reference has already been made (figure 30). The expression L Lf [1- ekt]?Ct (3)

takes account of this by the insertion of the term ct in which c could take a negative, zero or positive value as required. It has been found that equation (3) provides a reasonable fit with the experimental data. For the growth curve illustrated in

figure 34 least squares analysis using the Elliott 803 computer gives the following values of the constants in equation (3):

expt. temp. (o C) Lf x 103 CM k x 104 Sfen C x 1010 (CM s31) RP 15 30.0 OC 1.1 5-6 - 938




Successive regeneration of a given cell sample has been examined by Rosenbaum & Child (I967) for Ochromonas and by Randall et al. (I967), for Chlamydomonas. Experiments similar to the latter have been repeated utilizing the more accurate methods now available. These are of two kinds: (a) kinetics of regeneration, and (b) radioautographic studies in both the light and electron microscopes. Both series of experiments are still in progress and we shall report here on one experiment only in category (a).

00 0 4~~~~~~~~

E0 00 0 -

5X

0 200 400-------- --0 2000 3000 time (min)

FIGURE 34. 'Complete' regeneration curve for wild type (expt. RP 15), combining EM andLM results and allowing for shrinkage of embedded material.

0

it~() 0000I000 0 0 -~~~~~~~ ~~~0 0 0 000

0 0

1 st day 2nd day 3rd day 4th day

g v ~~~~~~Ia I I I 0 200 400 0 200 400

0 200 0 200 400 time (min)

FIGURE 35. Four cycles of deflagellation and regeneration in C. reinhardii at 25-5 ?C in NFM.

The regeneration of C. reinhardii (wild-type) has been examined at 25-5 'C over four successive cycles and the results are summarized in figure 35. The curves drawn have been determined (as above) by least squares analysis of the data in relation to equation (3). The corresponding values of the constants are collected together in table 2.




The fit between the experimental data and equation (3) provided by least- squares analysis is reasonably good, but should not be taken as the best and final representation in mathematical terms. The degree of fit is less good for small values of L, and for those (in the neighbourhood of t 100 min) where there is a sharp chanlge of slope in the regeneration curve.

TABLE 2. SUCCESSIVE REGENERATION OF C. REINHIARDII FLAGELLA

IN NFM AT 25-5 'C (EXPT. RS 14) (Values of constants in equation (3).)

regeneration Lf x 103 (cm) k x 104 (s-i) C x 109 (cM s1)

1 1-22 3.7 - 95 2 126 323 -1095 3 1-05 3 04 - 2 6

4 133 142 -1325

VI. DISCUSSION

As an interim report of work in progress on a wide but not wholly comprehensive front this paper necessarily leaves a number of important questions un- answered and others untouched. Three aspects not covered in these experiments are particularly important:

(i) Dynamic biochemical studies of regeneration with the aid of tracers aimed at discovering the site or sites of axoneme growth. Results obtained by Rosenbaum & Child (I967) led them to suggest the hypothesis of axoneme growth from the tip, but uneqiuivocal evidence of this is lacking. The difficulty is that a flagellum (apart from its recognizable subsidiary structures) embodies not only the axoneme, but the membrane and the matrix. At any moment of time during growth the matrix will contain subunit precursors and possibly species of free subunits prior to their attachment to the axoneme tubules. Consequently, both the matrix and the membrane may be rather indiscriminately labelled and thus obscure the record of the kinetics and sites of subunit attachment. A dynamic concept of the axoneme in which there is some continual turnover between it and its environment is possible. Growth at the tip plus turnover elsewhere would give a confused radioautographic picture.

(ii) Biochemical studies of flagella and cell bodies during the course of successive regeneration in NFM with the object of determining the, levels in the cell of (a) the free amino acid pool, and (b) flagellar protein, and (c) the transfer of protein from cell to flagellum, each as a function of time. Early experiments along these lines by Dubnau (I96I) were suggestive but not conclusive.

(iii) Direct intervention in regeneration by means of various possible effectors of growth and control. Dubnau (i96i) and later Rosenbaum & Child (I967) carried out experiments of this kind which it should now be possible to take further.

It will be conveniient in discussing the present experiments to deal first with genetical aspects; secondly with flagellar regeneration; and lastly with some questions of development and its regulation and control.




(1) Genetics Only a fraction of the eighty flagellar mutations now available have been

examined structurally and many of the new strains as well as the old remain unmapped. This situation is illustrated in figure 36 which presents the linkage groups of C. reinhardii as determined by Ebersold et al. (i962) with genetic informatioln on pf mutants as known then or as since supplemented by my colleagues. (It is understood that a new linkage map has now been worked out at

ac-14 c ac-76 arg-1 14b arg-2 pab-2 thi-3

I ,I, , .

14b I . . 10 2 2 4 15 30

ac-12 B

c 12b pf12 pf 18A-E nic-2 2a II L~~~~1L I I I

15 12 8 8

nf15A,B ac-28 pab c acl7 thi-2 0 6 20 6 < 0.1 24

thi-4 % 0

nic 11 c 4 pf2OA-E

1 7 30

31 ac-31a thi-8 c pf20 V I I . - I ,

4 10 20

nic-7 7a

Mnt C pf 14 24 12

VII1 1 _ Pf 17 ac-I thil C ac57 4 4 12-19 6

ac-51 pfl6 c a15 pf 13 19 8-15 15 15

ac 16 pf19A-E C pf2 ac-21 0 7 < -1 -5 25

FIGURE 36. The linkage map of C. reinhcardii based on data of Ebersold et al. (I962) and Warr et al. (I966), in which the main structural characteristic of some mapped flagellar mutants is indicated diagrammatically.

4 Vol. I73. B.




Harvard (Hastings, P. J., Levine, E. E., Coseby, E., Hudock, M. O., Gillham, N. W., Surzycki, S. J., Loppes, R. & Levine, R. P.), but this has not yet been published.) Four new linkage groups XII to XV have been added on one of which, XIII, a paralysed flagellar mutant pf 9 appears. Figure 36 serves to show the important points that the pf mutants so far discovered map on chromosomal genes and that a single gene is responsible for each particular mutant. The diagram also shows the association of structural modification in the flagella with certain genes. Information on these aspects is thus very much limited at present, but, again, the filling out of the picture is to a certain extent dependent only on the effort available. It is noticeable that mutations affecting the axoneme are apparently confined to modifications of the central pair of tubules (but see Miss McVittie's contribu-

tion, p. 59). The fact that the central pair is believed to be circumscribed by a helical thread or sheath illustrates the point that we do not know whether this has also been disrupted. While it is possible that some of the flagella-less, the stumpy and the short flagella mutants embody distortions and modifications of the outer nine pairs of the transition region TR, this is not yet a certainty. Moreover, the stumpy and flagella-less mutants cannot be mated and in consequence cannot be mapped or studied genetically. Thus while there is much to be done with existing mutants, there is also a great need for conditionally lethal mutants, perhaps of the temperature-sensitive type which in their own field the phage workers have found so useful. If such mutants can be found and if the structural modifications suggested by Miss McVittie's work are confirmed, the prospects of building up this side of the investigation are substantial.

The long flagellar mutants discovered by Miss McVittie (Randall et al. I967) may

be a consequence of a change in control at the genetic level. Short flagellar mutants could arise either from a similar cause or alternatively from a change in the

structure of the subunit. This could be reflected in their inability to assemble

correctly over substantial lengths. It is not clear at present which cause is operative. Since by definition a long flagellum is assembled to an abnormal length this

phenomenon seems more likely to be a function of control than of structure per se.

This argument, however, depends on the assumption that the axoneme of a long flagellum is normal in everything but length and this has not yet been extensively tested.

It is possible that the swollen flagellar mutants (table 1 and figure 23, plate 8) have membrane-defect tendencies. It is not know whether the swollen membrane

(itself a dynamic phenomenon) is a secondary (visible) manifestation of some other

event. (2). Flagellar regeneration

The analysis of flagellar regeneration kinetics has been stressed as a useful

approach to the understanding of the mechanisms of growth and its control. As

we have seen in ?V, the length of the flagellum (as the mean of a sample popu-

lation) at a given temperature can be measured reasonably accurately. Parallel

studies in the electron microscope have shown that in C. reinhardii there is no lag




period. Moreover, the successive regeneration of flagella without nitrogenl replenish- ment from the medium indicates that the cell has substantial reserves. As indicated above, it will be valuable to learn the nature and quantity of these reserves4in terms of amino acids and proteins as the regeneration (single or multiple) proceeds. The experiments of Dubnau (I96I) on changes in the amino acid pool of Ochromonas danica were carried out in a nitrogen-free medium which was also lacking in biotin to prevent cell division. A drop in pool level of about 15 % was always observed during the first 40 min of regeneration, and this was followed by a gradual recovery to the control level. From the reproducibility of this result the author concluded that a utilization of free amino acid takes place during the early stages of regeneration. Rosenbaum & Child (I967) have carried out more elaborate experiments with Ochromonas and Euglena using [311]leucine, and have investigated the effects of cycloheximide, an inhibitor of protein synthesis, on these organisms. It was shown that there was much more incorporation (75 and 50 % respectively) of [3H]leucine into regenerating flagella than into fully grown flagella by the end of the regeneration period. It has already been pointed out that difficulty in interpretation arises when the data refer to the whole flagellum and the focus of attention is what has been incorporated into the axoneme. Until the axoneme can be separated from its sheath and the other contents of the flagellum, which may include non-specific cytoplasmic proteins, the questions of turnover and the site of growth will remain unresolved.

(3). Problems of development, its regulation and control

So far no coherent picture of the basic mechanisms of mnorphopoiesis in the flagellar apparatus has emerged, nor seems likely to do so in the near future. I shall now refer to a number of the more significant aspects of the problem.

(a) Organelle DNA

The existence of DNA at the sites of basal bodies has not yet been demonstrated for Chlamnydomonas, but only for Tetrahymena (Randall & Disbrey I965) and Paramecium (Smith-Sonneborn & Plaut i967). Genetic evidence shows that the structural genes of the FA are chromosomal. This does not preclude the existence of 'basal body' DNA in the form of copies of the relevant nuclear genes, nor does it preclude the possible presence of a distinct satellite DNA concerned with control mechanisms. Without new facts there is little point in further speculation.

(b) The natural development in the zygote

It is convenient to consider the known sequence of events that occurs in the maturation of the zygote and as reconstructed diagrammatically from electron micrographs in figure 37. First there is the development of the basal body and the transition region. Then comes the simultaneous formation of the axoneme and its enclosing membrane. The equally simultaneous cessation of growth in the axoneme and its lumen signifies the imposition of a control mechanism. The development

4-2




(0) (iv)

(vii)

TR?

BB cw

(Viiif ) (ix) (x)

CF

(xi)

(Xii)

FIGURE 37. Diagrammatic reconstruction of suggested course of morphopoietic events in the development of the FA of C. reinhardii subsequent to the disappearance of the basal body in the zygote:

(i) Axial tubule of cartwheel structure. (ii) Attachment of ninefold sheet structure. These two stages represent one inter-

pretation of the evidence. It is possible that the radial structure of (ii) to (vi) should be fibrous and not sheetlike as shown.

(iii) Attachment of nine singlets (C tubules) to radial structure of (ii). (iv) Possible stage in attachment of A and B tubules to (iii). A and B tubules are

continuous with the outer nine pairs of the external flagellum. (v) Stage (iv) increases in length. This and the previous stage could be simultaneous. (vi) Transverse view of (v). (vii) Attachment of basal body to plasma membrane at distal extremity. (viii), (ix) and (x) Give views of the completed basal body and transition region

structures. (xi) Indicates the initial protrusion of the plasma membrane as the A and B tubules

extend and transition region TR in (ix) is completed. (xii) Early stage of growing flagellum with central pair, side-arms and radial spokes

are hypothetically presumed to be attached during this period.



Flayellar apparatus as a model organelle 53

of the basal body is particularly difficult to deal with in other than descriptive terms, because no techniques have yet been developed for isolating it in pure form. It is perhaps significant that the microtubules of the basal body are developed in situ in the cell cortex and do not appear to migrate to their final positions, as appears to happen in ciliated mammalian tissues (e.g. Dirksen & Crocker i966; Sorokin I968). Microtubules, unorganized into more complex structures, have now been observed (see ?1 above) in the bodies of many non-flagellate and non- ciliate cells. The ability to convert polypeptide chains into suitable subunits is not therefore universally restricted to the cell cortex or flagellar lumen.

There is always some element of doubt in the assumption that all parts of a biological structure as necessarily significant for its ultimate function. As pointed out by Hookes et al. (I967), some structural component may be required only for a sequential development; it may or may not be subsequently discarded. As will now be seen both basal body and transition region contain features essential to the structure of the external flagellum.

Basal body development precedes that of the flagellum. In figure 37(i) the formation of basal body C tubules is depicted as taking place separately from the development of the cartwheel structure of figure 37 (ii). It would be interesting to know the precise sequence of these events. A priori it seems unlikely that the C tubules themselves would have symmetry properties imposing a ninefold axis on the system. It is therefore reasonable to suggest that the purpose of both the basal body 'cartwheel' and the 'star' of the transition region is to impose the ninefold symmetry and structure on the outer flagellum. It is impossible to guess why such a ninefold structure should be essential to its proper function. In figure 37 the basal body 'cartwheel' has been shown as a system of radial sheets. Strictly speaking this cannot be deduced from the evidence available. The structure may be fibrous rather than laminar. For the present purpose this does not matter, since we are here chiefly concerned with symmetry.

In many organisms the central pair tubules are joined to the TR structure, usually by a globular unit. Although such connexion is not visible in Chlamydo- monas, there can be little doubt that an additional function of the transition reoion is to position these tubules.

(c) Regulation and control

It is not yet understood why the structures of the FA are of the form that is observed, nor is it understood how these structures are controlled. The external flagellum is the obvious component for study and measurement, as the regeneration experiments have already shown. Our final remarks will deal with this question of control.

It is first of all important that the flagellar apparatus is cortical. Even the flagellar roots, of which there are 16, are confined to this region (see Ringo I967,

figures 27 and 28). A substantial fraction of the cell volume is occupied by nucleus, chloroplast and pyrenoid. It seems inescapable that during FA development the




cytoplasm will contain amino acids, precursor protein chains, and free subunits of the appropriate kinds. A regulatory system in the form of feedback inhibition (as found in the studies of Novick & Szilard (I954) and Umbarger (1956, I96I)) would clearly act within the cytoplasm. Alternatively, a genetic repression control system (Jacob & Monod I96I, I963) would act from the DNA within the nucleus or from DNA suitably disposed in the cytoplasm.

The cortical site of the organelle, and the more or less precise positions of the external flagella, suggest that the cell membrane system is suitably differentiated for this purpose. Further it seems possible that the flagellar membrane could also be the site of enzymes that convert precursor protein chains into subunits. A product of this conversion (e.g. a small peptide) could inhibit further formation of subunits and so regulate the length of the flagellum. It is therefore most important to determine the sites of synthesis and of chain-to-subunit conversion for the axoneme. Presumably the basal body and transition regions are wholly organized in the cytoplasm. A further argument about length regulation comes from the multiple regeneration experiments. The cell is capable (in NFM) of making a total length of flagellum equal to 2nL where L is the length of one 'normal' flagellum. But it cannot produce this over-all length in units greater than L after each amputation. The inhibition of growth is thus a functioln of the subunits made and incorporated into the axoneme. The over-all limiting reaction as determined by the regeneration experiments is roughly monomolecular: whatever reaction it represents proceeds to approximately zero concentration (there will of course be several such reactions of similar characteristics). This could most obviously come about by inhibition of subunit formation within the flagellum itself. Amputation of flagella removes axoneme and inhibitor and regeneration is thus allowed to begin. Jacob & Monod (i 963) have pointed out, however, that 'the reaction catalysed by allosteric enzymes very often if not as a rule obeys multimolecular rather than monomolecular relations with respect to both substrate and inhibitor'. Obviously the question of length regulation alone cannot be settled by paper arguments. The solution to this problem requires a number of new and precise experiments on the flagellum and the cell relating to the synthesis of proteins and to the formation of subunits, both considered as rate processes, coupled with the search for inhibitors. The existence of mutants with flagella of abnormal length gives a valuable genetic approach which should be exploited in the search for the basic factors of length regulation. The corresponding problems of the other cortical components of the FA are much more difficult and a solution to them is a somewhat distant prospect. Two other aspects of length regulation should be mentioned. Although the above suggestion for the origin of regulation gives a significant role to the flagellar membrane or lumen it does not explain how the membrane itself is also length limited. There is probably some common factor in these two intimately associated systems, perhaps in the nature of one of the proteins or, alternatively an inhibitor, which may become apparent when experiments to test the main proposal are carried out.




Lastly, there is the question of control in the twin flagella, i.e. the FA as a whole, emphasized by experiments on partial or asymmetric deflagellation. These remind us that the two flagella of C. reinhardii are not in general exactly equal in length but only approximately so. The tendency towards precise equalization is probably counterbalanced by small random changes in the extremely localized biochemistry of each flagellum. Nevertheless, the tendency towards equalization is a dominating one. This is shown by the asymmetric deflagellation experiments, which also bring to our notice a latent mechanism of regression. If, for example, -half of one flagellum is amputated the other begins to shorten until the two are roughly equal in length. Regeneration then proceeds until both have grown to 'normal' length.

Such imposed dissymmetry evidently disturbs the biochemical balance between the two flagella which has to be at least partly restored before the normal regenerative mechanism becomes the major factor. Complete restoration of balance does not appear to be necessary, as it has been observed that the shorter flagellum may begin to regenerate before the longer ceases to regress. The deliberate produc- tion of dissymmetry thus reveals an additional complexity and interrelationship between the two external components of the twin system not evident in either the normal mature organelle or in the completely deflagellated (but still 'balanced') system.

In more general terms, it will be necessary to discover what degree of turnover exists under the various conditions of regeneration implied above both in the flagellar membrane and the axoneme. This should help to decide between the following questions: (1) Is the length of flagella at any time t the net result of competing regenerative and regressive mechanisms, both active at all times, but only approximately in balance when the flagellum is 'fully grown'? Or (2) Is only one of these processes active under given conditions and the other suppressed? It will not be sufficient to examine turnover in the flagellum as a whole; it should be assayed for the three major components: lumen membrane, axoneme and matrix.

The flagellar apparatus of C. reinhardii is seen to be a highly integrated complex system composed of structurally, biochemically and genetically distinct components. Knowledge is increasing slowly but steadily. Although new problems are posed at every stage, it is now possible to have some insight into what is involved and to conclude that research on this organelle is an important route to the understanding of morphopoiesis and growth.

APPENDIX

Temperature control apparatus used in fiagellar regeneration experiments

BY H. R. MUNDEN AND P. H. PREST

Figure 38 illustrates diagrammatically the type of apparatus used to control temperature in the regeneration experiments of ?V. The centrifuge box on the



a discussion on cytoplasmic organelles || the flagellar apparatus as a model organelle for the study...

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