Control of Flagellum Number in Naegleria Temperature Shock Induction of Multiflagellate Cells

jf. Cell Sci. 7) 463-481 (1970) 463 Printed in Great Britain

CONTROL OF FLAGELLUM NUMBER IN NAEGLERIA TEMPERATURE SHOCK INDUCTION OF MULTIFLAGELLATE CELLS

A. D. DINGLE Department of Biology, McMaster University, Hamilton, Ontario, Canada

SUMMARY This paper describes the production of supernumerary flagella by flagellates of Naegleria gniberi exposed to sublethal temperature shocks during the amoeba-to-flagellate transformation. When transformed at any constant temperature below 34 CC, cells of N. gniberi strain NB-i may develop from 1 to 4 flagella, but biflagellate cells predominate and the average number of flagella per cell in these populations is approximately 22. Populations exposed to a 38 °C temperature shock during transformation develop about twice as many flagella as controls (average number of flagella per cell is approximately 4's). The individual response of cells is extremely heterogeneous: some develop no more than the normal 2 flagella, whereas others develop as many as 18. At least half the cells produce 5 or more flagella, as opposed to fewer than 1 % with 5 or more flagella in control populations. Multiflagellate cells and populations are normal in appearance, apart from the excess flagella and resulting disorientation of the normal swimming pattern. The supernumerary flagella, their basal bodies and the rhizoplast (which is also occasionally doubled) are indistinguishable from those of normal cells. Maximal production of flagella occurs over the narrow temperature range from 37-5 to 385 °C and is related to the duration of exposure to a given temperature: optimal flagellum induction is obtained following a 45-50 min exposure to a 38-2 °C temperature shock. After approximately 1 h the flagellates revert to amoebae, losing both normal and supernumerary flagella. Populations of these amoebae followed through 2 more cycles of retransformation and reversion developed the normal number of flagella (22). Clones isolated from multiflagellate populations were identical to normal populations, developing approximately 2-1 flagella per flagellate at normal temperatures and 45 flagella per flagellate when temperature shocked again.

INTRODUCTION Both the number and the distribution of cilia and flagella on eucaryotic cells are subject to enormous variation. For example, flagella are often found singly as on many protistan cells, on virtually all sperm cells and on the retinal rods of photoreceptors. They are frequently paired, as on other protistan cells, on epithelio-muscular cells of coelenterate gastroderm, and on cells of the rat adenohypophysis. Several or even thousands of flagella may be present on a single cell, as in the case of polymastigote flagellates, ciliates, sperms of ferns and cycads, and in the ciliated epithelia of both vertebrates and invertebrates. This extreme variability in the number and distribution of flagella raises intriguing questions about cellular differentiation and regulation at the organelle level. One such question concerns the nature of the controls which direct the development 464 A. D. Dingle of precisely one flagellum on mammalian spermatozoa (Fawcett, 1958), two flagella on cells of mammalian adenohypophysis (Wheatley, 1967), and hundreds of cilia on the ciliated epithelium of mammalian trachea (Dirksen & Crocker, 1966) or lung (Sorokin, 1968). In multicellular organisms these are developmental controls in which each different length, function and distribution is expressed against a constant genetic background. In the case of many unicellular organisms, so precise is the genetic control of flagellum number and distribution that these parameters often constitute important criteria for taxonomic characterization. Many examples come to mind, such as Monas and Polytoma with 1 flagellum, Chlamydomonas and Bodo with 2 and Tetramitus, Hexamitus and Octamitus with 4, 6 and 8 flagella respectively. Consider also the following comment on the classification of chrysomonad flagellates (Hall, 1953): 'on the basis of flagellar equipment, four families have been created: Chromulinidae, with one flagellum; Syncriptidae, with two equal flagella; Ochromonididae, with one long and one short flagellum; and Prymnesiidae, with three flagella.' Despite many such examples of rigorous controls over flagellum number, there have been repeated observations of cells with unusual numbers of flagella, for example mammalian sperm tails with double axonemes (Fawcett, 1961), and the atypical multiflagellated sperms of the snail Viviparus (Gall, 1961). Furthermore, the cells of many species routinely have variable numbers of flagella: newly flagellatedswar m cells of the myxomycete Didymium nigripes possess only one flagellum but after several hours may develop a second (Kerr, i960). More impressive is the transition in which virtually all cells of mammalian foetal lung—whether fibroblast, chondrocyte, myo- cyte, mesothelial cell or epithelial cell—initially develop single 'primary' or (9 + 0) cilia. Later, only those cells of the maturing lung which differentiate into the epithelial border develop 250-300 basal bodies and cilia (9 + 2) apiece (Sorokin, 1968). The flagellateso f Naegleriagniberi have been described as possessing predominantly 2 (Alexieff, 1912; Wilson, 1916) or predominantly 4 flagella(Willmer , 1956). Normal flagellate populations of the NB-i strain are composed of cells bearing from 1 to 4 flagella and, very rarely, exceptional cells are seen with 5 or more (Fulton & Dingle, 1967; reviewed in Fulton, 1969). During an investigation of temperature effects on the rate of Naegleria transformation it was noticed that among the reduced numbers of cells which developed flagella at sublethal temperatures (approximately 40 °C), there was a significant increase in the proportion of cells which produced 3, 4 or more flagella. At that time it was realized that the amoeba-to-flagellate transformation of Naegleria presented a near unique system with which to study the development of the centriole-like basal bodies in cells which have no visible centriole precursor. This is a particularly intriguing possibility in the light of the commonly accepted generaliza- tion that centrioles and basal bodies develop only in association with pre-existing centriole-like templates. However, because of their small size and limited number, centrioles are not easily visualized in cells, either by light or electron microscopy. Some means of either enhancing the resolution or increasing the number of centrioles and basal bodies is therefore a prerequisite for the elucidation of the developmental sequence by which these organelles arise. This paper presents such a method, based on a sublethal temperature shock, which Control of flagellum number in Naegleria 465 induces the development of approximately double the normal complement of basal bodies and flagella in transforming populations of Naegleria. The appearance and behaviour of the multiflagellate cells and their flagellar apparatuses are compared with those of normal biflagellate cells, and the longevity and heredity of the excess flagella and basal bodies are discussed in the light of the supposed continuity of centrioles and basal bodies from one cell generation to the next. Subsequent reports (A. D. Dingle, in preparation; C. Fulton & A. D. Dingle, in preparation) will describe the effects of environmental variables on the induction of excess flagella, and the use of these 'hairy' populations in studies of the development and continuity of basal bodies in Naegleria. A preliminary report of this work has been published (Dingle, 1967).

MATERIALS AND METHODS Petri-dish cultures of Naegleria gruberi strain NB-i were grown at 34 °C on nutrient agar (NM) in association with the bacterium Aerobacter aerogenes, as described by Fulton & Dingle (1967). Cells from early stationary-phase cultures were suspended in 0-002 M tris (hydroxymethyl) aminomethane (tris) buffer, pH 74, washed free of bacteria by differential centrifuga- tion using the method C described by Fulton (1969), and resuspended in tris at final cell densities of 5-10 x io5/ml. Aliquots of 15-20 ml of these suspensions were dispensed into unstoppered 125-ml Erlenmeyer flasks and shaken at 100 2-5-cm strokes per min under various temperature regimens as described in the Results section. Particular care was taken to control water baths to within ±o-i °C of any reported experimental temperature. Cell densities were determined by diluting aliquots of the suspension into a large volume of electrolyte (04% NaCl), agitating vigorously and counting in a Model F Coulter counter at threshold 20 (attenuation 2; aperture current 64). Mean cell volumes were likewise determined by threshold sizing with the Coulter counter which had been previously calibrated in cubic micrometres per threshold division (Sheldon & Parsons, 1967) against direct measurements made on 1000 NB-l cyst diameters using a Nikon filar micrometer eyepiece. At intervals, samples of transforming cells were fixed and stained in Lugol's iodine at a ratio of 4 drops cell suspension to 1 drop fixative. Using phase-contrast optics at 500 x magnification, the percentage of flagellated cells (%F) was determined for samples of 100 cells, and counts of the average number of flagella per cell (f/F) were made on samples of at least 300 flagellates. Light-microscope photomicrographs of iodine-fixed flagellates were taken using phase- contrast optics and Ilford FP-4 film in a Nikon EFM semi-automatic camera attachment. For electron microscopy, cell suspensions were initially centrifuged lightly into loose pellets, then resuspended and fixed for 30 min at room temperature in a solution containing 3 % glutaral- M dehyde, 0002 M CaCl2, and 001 M NaCl, in 0-005 tricine (iV-£m(hydroxymethyl)methyl- glycine) buffer, pH 74. The cells were centrifuged into a pellet again, rinsed briefly with tricine buffer, and post-fixed in cold 1 % osmium tetroxide for 30 min. The pellets were then stained for 1 h in cold saturated aqueous uranyl acetate, dehydrated rapidly through ethanol to pro- pylene oxide and embedded in Araldite 502 (Coulter, 1967). Grey and silver sections cut with a diamond knife on an LKB Ultrotome were spread with chloroform vapours, picked up on uncoated 300-mesh grids and stained 1-2 min with Reynolds' (1963) lead citrate. Observations and micrographs were made with an RCA EMU-3H and a Philips EM 300 microscope.

RESULTS Induction of excess flagella by heat shocks When transformed according to the standard transformation experiment of Fulton & Dingle (1967), that is, grown at 34 °C and washed and transformed at 25 °C, flagellate populations of N. gruberi strain NB-i have an average of 2-2 flagella per 466 A. D. Dingle flagellate cell (f/F). Approximately 13 % of the flagellates have 1 flagellum, 65 % have 2 12 % have 3 and 8 % have 4 flagella; fewer than 1 % have 5 or more flagella.I f the cells are transformed at any constant temperature in the range 10 to 34 °C, a similar distribution of fjF ensues. However, as the temperature is raised beyond 35 °C, transformation becomes somewhat erratic and incomplete, and among those cells which do transform one notices a significantly higher proportion of cells with 3, 4 and more flagella. At higher temperatures fewer cells are capable of transforming, until fewer than 10% of the cells become flagellated if left at 38 °C.

100 r 300

"5 38 °C "o 200 Temp, shock 470 f/F

3 z 100

01- 30 60 90 120 150 123456 12345678 910 Minutes after suspension Flagella per cell Fig. 1. Induction of excess flagella in Naegleria by high-temperature shock. Amoeba were harvested from 34 °C-grovvth plates, washed and resuspended in tris buffer at 38 °C. One aliquot of cells was immediately transferred to 25 °C while the other was maintained at 38 CC until 50 min after initial suspension. Fig. 1 A. The transformation to flagellated cells in the 25 °C control (O) and 38 °C temperature-shocked (•) samples. Each point is the number of cells with flagella per 100 Lugol's iodine-fixed cells. Fig. 1 B. Frequency histograms showing the number of flagella on 500 flagellates from fully transformed samples (tl2o min) of control and temperature-shocked cells.

If these heat-shocked cells are returned to 25 °Cj following a period of approximately 50 min at 38 °C, more than 90% of the population transforms normally after a 10-20 min delay (Fig. IA). However, a fundamental change occurs such that the average number of flagella is roughly doubled—from 2-2 to 4-6 fjF (Fig. IB; also compare Figs. 6 and 7). The range oif/F increases from 1-4 to 1-10 and (rarely) cells with as many as 18 flagella are seen. The overall impression one gets from light- microscopic observations of these cells (Fig. 7 and inset) is of flagella bristling from the surface at all angles like so many hairs, and these cells and populations with excess numbers of flagella are referred to as 'hairy'.

Nature of hairy cells Apart from their great excess of flagella,population s of hairy cells are similar to those of normal flagellates (Figs. 6, 7). Temperature-shocked flagellates tend to be less typically ' flagellate-shaped' than untreated cells. They are somewhat rounded in Control of flagellum number in Naegleria 467 outline and less fusiform than the idealized Naegleria flagellate,althoug h many hairy cells in the population do achieve the typical flagellatemorphology . Deviations from the normal shape fall into 2 major categories; either the cells are nearly spherical or they are somewhat triangular in outline. These altered shapes can be roughly related to the distribution of additional flagella. An apex is generally found at the site of any cluster of 3 or more flagella. Flagellates which are spherical more often have 1 or 2 flagella extending from several sites about the entire periphery of the cell, although some spherical cells are also seen with large clusters of flagellaprotrudin g from a single site. Obviously the distribution of flagella on these cells is extremely irregular and no definitive description of their placement can be given. Nevertheless, certain consistent arrangements are seen: flagella are most often present in pairs or in clusters of 3, 4 or 5; they are generally concentrated at one end of the cell and the nucleus is usually found close by the major cluster of flagella. This grouping of flagella in hairy cells is consistent with previous observations of normal flagellates (Schuster, 1963; Dingle & Fulton, 1966) in which the flagellar basal bodies were shown to connect with splayed branches of a rhizoplast by a short palisade of microtubular filaments. The threadlike rhizoplast then runs to the edge of the nucleus where it is contiguous with but apparently does not penetrate the nuclear envelope. The electron micrographs (Figs. 8, 9) illustrate this connection of rhizoplast and basal bodies in hairy flagellates. Fig. 8 is interesting inasmuch as 2 separate pairs of basal bodies appear to be attached to 2 distinct rhizoplasts. Usually, when a cluster of flagella is seen at one terminus of the rhizoplast, other basal bodies and flagella which may be seen in the same section are devoid of connections with that rhizoplast, although cells with more than one rhizoplast are often seen in populations of hairy cells. It is further apparent from Figs. 8-10 that additional flagella induced by the temperature shock are normal in their fine structure, and that each new flagellum has at its base a typical centriole-like basal body. All the flagella seen in populations of hairy cells appear to be normal, not only as judged by light and electron microscopy, but also in their characteristic flagellar beating. Hairy cells on the other hand do not swim normally. The irregular placement of numerous flagella about the cell surface results in an unusual motion in which the hairy flagellates tumble randomly through the medium rather than swimming, as normal flagellates do, in tight spirals along relatively straight paths. The visual impression one gets from scanning fields of temperature-shocked and control flagellates is that hairy cells are somewhat larger than normals. If true, this observation might suggest reasonable ways in which hairy cells could arise in the population, e.g. the cells could have continued to grow after suspension for transformation, but were unable to divide at the elevated temperature, or cell fusion could be creating giant cells with excessive amounts of nuclear, cytoplasmic and, particularly, flagellar materials.. The following observations indicate that neither of the above suggestions can be valid. (1) Coulter counts of cell density repeated at 5-min intervals from initial suspension throughout the entire transformation sequence remained roughly constant in both control and temperature-shocked populations. Had there been any appreciable fusion of cells, counts of cell density would have dropped proportionately. (2) Mean cell

30 C E L 7 468 A. D. Dingle volumes of temperature-shocked Naegleria were likewise measured at 5-min intervals throughout the transformation by means of threshold sizing with a Coulter counter. There was no increase in cell volume corresponding either to cell growth or to fusion. In fact, the mean cell volume of the hairy flagellates decreased by approximately 11 % 3 3 from tw min (1055 /tm ) to t120 min (934 /tm ). (3) The excessive numbers of flagella are not correlated with any increase in nuclear size or number. Amoebae and temperature-shocked flagellates were fixed (4:1) in saturated HgCl2, and the number of nuclei per cell was counted for samples of 1000 cells using 400 x phase-contrast optics. Nuclear diameters were measured in the same samples using a Nikon vernier eyepiece micrometer. Approximately 1-2% of amoebae washed from NM growth plates were binucleate, and only 1-5 % of the hairy flagellates had 2 nuclei: none had more than 2 nuclei. Similarly, the average nuclear diameter was 3-14/(111 in freshly suspended amoebae, 2-89/tm in control flagellates with 2-12 f/F, and 2-93/6111 in temperature-shocked flagellates which had 4-21 f/F. Clearly the increase in flagellum number cannot be related to any process of cell or nuclear growth, or cell fusion resulting from the temperature treatment.

Quantitative evaluation of hairiness Comparison of histograms showing the number of cells with a given number of flagella readily illustrates the marked differences between control populations and temperature-shocked cells (Fig. 1 B). However, in attempting to define optimal conditions for inducing excess flagella, one must have a better measure than the sub- jective impression left by comparison of histograms. Since the optimal treatment may be defined as that which produced the maximum number of flagella, the parameter fjF (average number of flagella per flagellate) is particularly appropriate. How- ever, because transforming cells do not respond uniformly to temperature shocks— some develop the usual 2 flagella and others develop from 1 to 16 supernumerary flagella—comparisons of f/F are inadequate by themselves. Two populations, one with many cells having 3 and 4 flagella, and the other with very few cells having excess flagella but those cells having 10-20 flagella, could give similar fjF values. Such a situation can be partially indicated by including the range of flagellum number along with the average number of flagella,althoug h discontinuous distributions can also create misleading impressions about populations based on range of flagellum number. A second useful measure of the extent to which a particular treatment has induced excess numbers of flagella is the percentage of flagellates which have 5 or more flagella, % (5 +). Since, in the normal 25 °C transformation, cells with 5 or more flagella are rarely encountered (approximately 1 %), and in temperature-shocked populations % (5 +) may reach 50 or more, this provides a quick and simple measure of hairiness. Another useful indicator of the temperature effect is the percentage of flagellates having 1 or 2 flagella, % (1,2). Approximately 80% of the cells transformed at 25 °C will have 1 or 2 flagellawherea s fewer than 10% of flagellates obtained after effective temperature shocks will have so few flagella. One advantage of this parameter over % (5 +) is that it expresses not only the proportion of cells which have unusually high Control of flagellum number in Naegleria 469 numbers of flagella, but also shifts within the population from 1 and 2 to 3 and 4 flagella. In Fig. 2 the effects of a 50-min period, at temperatures from 340 to 40 °C are plotted as//JP, % (5 + ), and % (1,2). In each case the measured effect is seen to be greatest within the narrow temperature range 37-5-38-5 °C. Although each of these parameters is measuring a different aspect of the flagellum distribution within the populations, their parallel response to various temperature-shock protocols indicates a basic similarity. The effectiveness of any flagellum-inducing treatment is therefore routinely expressed in terms oif/F, % (5 + ) and % (1,2).

45 ~ Average number of 0/ cells with 5 % cells with /o flagella per cell or more flagella 1 or 2 flagella 8 0 O 40 40 - fp 20 3-5 30 - / 0 40 6 0 30 20 - 60

25 10 - 80

0 /oj ' 38-2°cV 38 1 °C 20 0 -e—e 100 0 O o 1 1 1 , 11 1 . . . . I 34 35 36 37 38 39 40 34 35 36 37 38 39 40 34 35 36 37 38 39 40 Temperature (°C) Fig. 2. Induction of excess flagella by a 50-min heat shock at temperatures ranging from 340 to 40 °C. Results are expressed as average number of flagella per flagellate (J/F), % of flagellates bearing 5 or more flagella % (5+), or % of flagellates with 1 or 2 flagella % (1,2) on the ordinates (note that % (1,2) is inverted). Each point represents the average value for 300 cells counted. All 3 parameters used to express the hairiness phenomenon illustrate a maximum effect in the narrow temperature range of 37'5-38'5 °C

It has been demonstrated by Fulton & Dingle (1967) that systematic errors in counts of flagellum number occur due to a problem of 'cell geometry', as flagellawhos e length is less than one half the cell diameter occasionally lie hidden beneath the cell. Such errors are negligible in counts of flagella on mature flagellates from standard transformation. However, in the case of hairy cells, it is apparent that many of the additional flagella do not attain the normal length and flagellum counts must be somewhat inaccurate. To evaluate the extent of such errors in flagellum counts, samples of 500 normal and 500 heat-shocked cells were counted, then recounted after careful rolling revealed all possible flagella. Cells fixed and stained with Lugol's iodine were initially counted under slightly elevated coverslips, using phase-contrast optics at 30-2 470 A. D. Dingle 400 x magnification. After each cell was scored for flagellum number, it was rolled about by means of gentle, controlled pressure exerted on the coverslip by a Brinkman micromanipulator, and all additional flagella were recorded. It is apparent from the histograms of Fig. 3 that there are appreciable inaccuracies in flagellum counts made on populations of hairy cells: in particular, the counts of cells with 2 and 3 flagella are exaggerated at the expense of counts of cells with 6 or more flagella. In terms of the average number of flagella per flagellate, this is expressed as a decrease from 5-25 to 4-68 f/F, which means that routine counts of f/F underestimate the true values by

400 r

100 -

6 7 12 3 4 5 7 8 Number of flagella per cell Fig. 3. Frequency histograms showing changes in flagellum counts before ( ) and after ( ) cell rolling. The number of flagella per cell was counted, then recounted after carefully rolling with a micromanipulator. These histograms represent counts made on 500 Lugol's iodine-stained flagellates obtained from a 'standard' transformation and 500 flagellates from a 38-2 °C (0-50 min) heat-shock transformation experiment. approximately 11%. In the case of the 25 °C normal flagellate population, the f/F value increased by approximately 6%, from 2-30 to 2-44 upon rolling. Thus the magnitude of the counting error increases as//F values increase. Since the hairiness phenomenon is not a subtle change but a 2-fold increase in f/F, and since these errors are conservative, leading to an underestimate rather than an overestimate of the effect, routine counts of flagellum number are made without resorting to the laborious and time-consuming rolling procedure. Another possible source of error in measuring fjF lies in the fact that temperature- shocked flagellates begin to revert to amoebae approximately 50 min after the population becomes completely flagellated. Since not all flagella on hairy cells reach their mature lengths at the same time, it is conceivable that the earliest transforming cells could begin reverting (losing flagella)befor e all cells in the population developed their Control of flagellum number in Naegleria 471 maximum number of flagella. In order to determine the time or times at which fjF values are maximal, counts were made on 7 samples of 300 cells fixeda t io-min intervals throughout the entire 60-min period during which the population was at least 90% flagellated. In this particular experiment the fjF was 4-19; the standard deviation for the samples was 0-21 and each sample within that time interval fell within the 95% confidence interval for the overall mean. Thus, there is no significant variation in f/F values measured over the entire 60-min sampling interval during which the population was 90% or more flagellated (see also Fig. 5). Nevertheless,//^ values are routinely obtained by counting the flagellum distribution for 300 cells, 100 at each of 3 successive io-min intervals past the point at which the population first becomes 90% flagellated. This repetitive sampling procedure has the advantage of immediately revealing any inconsistencies in counts which could result from various sampling errors.

Time and temperature for maximal hairiness induction It has already been demonstrated (Fig. 2) that the critical factor in the flagellum induction phenomenon is exposure to sublethal temperatures during transformation. Cells which, are heat-shocked from t0 (suspension) to ti0 min develop the greatest excess of flagella in the very narrow temperature range 37-5—38-5 °C. The length of exposure of transforming cells to the heat shock is also crucial to the attainment of the

3 5

10 20 30 40 50 60 Minutes at 38 °C Fig. 4. Optimal duration of exposure to heat shock for inducing excess flagella in Naegleria. Cells were suspended from growth plates, washed and resuspended in 380 °C tris, then returned to 25 °C at io-min intervals to complete transformation. Each point represents the mean and standard deviation of 5 independent experiments, in each of which f/F was determined by counting 300 flagellated cells. maximum number of additional flagella. These time and temperature relationships will be described more thoroughly in a subsequent paper on the hairiness phenomenon in which the 'critical temperature-sensitive interval' will be precisely defined and related to other crucial events in the transformation process (A. D. Dingle, in preparation). For purposes of the present discussion it will suffice to point out that the optimum time of exposure varies with the exact heat shock temperature. Longer exposures are required to produce the maximum number of flagella at higher temperature, e.g. 472 A. D. Dingle at 35 °C the optimal time of exposure is 30 min, at 37 °C it is 35 min and at 39 °C it is 60 min. Fig. 4 illustrates the increase mf/F values obtained with increasing exposure to 38-0 °C-temperature shocks. Relatively few additional flagelladevelo p in the populations until the duration of exposure reaches 30 min. Thereafter a dramatic increase occurs up to a plateau value of approximately \-if\F for exposures from 40 to 50 min. Longer exposures then decrease the average number of flagella per cell in 38 °C- shocked populations until, beyond 60 min, transformation becomes heterogeneous and incomplete. Consequently, my routine protocol for producing flagellate populations whose average number of flagella per cell is at least double that of controls is to wash the suspended 34 °C-grown amoebae in tris buffer at 38-2 ± o-i °C for 45 min, and to return them to 25-0 + 0-1 °C to complete transformation.

Fate of supernumerary flagella on hairy cells Centrioles are generally considered to be permanent and heritable cell organelles, replicating at an early stage of division and passing from mother to daughter cells at each generation. In view of the structural and functional homologies between centrioles and the basal bodies of flagella, it became of interest to determine if the excess

Table 1. Comparison of transformation and average flagellum number of 12 clones isolated from a population of temperature-shocked hairy flagellates

Standard 25 °C transformation 38 °C temperature shock

Sample T50 Mp f/F MF fIF

Original 76 97 2-12 88 97 4-5° suspension Clone 1 75 93 2-12 82 95 4-11 2 72 95 217 80 95 4 "44 3 73 94 2O2 84 86 4-08 4 79 97 2-O7 89 100 4-58 5 7O 94 2-O9 84 98 4-46 6 73 100 2-15 82 99 4-52 7 72 99 2IO 79 100 4-25 8 75 96 2 OO 81 95 4-21 9 71 94 2-30 78 97 4-55 10 71 95 2-OS 80 98 4-28 11 71 98 2O7 81 96 4-53 12 69 98 2l6 80 96 452 flagella and basal bodies induced by heat shocks were inherited by succeeding generations of Naegleria, or whether these were simply diluted out of the population at successive cell divisions. To investigate the subsequent fate of the additional flagella, a population of heat-shocked flagellates was Coulter counted, diluted to give approximately 20 cells per plate and plated with Aerobacter on clone plating medium PM, following the procedure of Fulton & Dingle (1967). Twelve clonal isolates were selected in this manner from a sample of hairy flagellates in which 87 % of the cells had 3 or more flagella {fjF = 4-35). Two samples of each clone were replated with Aerobacter Control of flagellum number in Naegleria 473 on normal NM growth plates; one sample was then transformed under standard conditions at 25 °C throughout, while the other was subjected to the usual 0-45 min temperature shock at 38-2 °C. The results of this experiment (Table 1) indicate that, in terms of transformation kinetics (7^,, and MF) and average flagellum number (f/F), populations of cells grown from each of the clones are indistinguishable from one another and from the original suspension. If it may be presumed that any extra basal bodies present in transforming cells would express their presence by organizing and extruding flagella, then it is clear that hairiness is not heritable, that the excess basal bodies have been diluted out of the cell populations in subsequent generations, for no single clonal suspension had an

100 r -1 50

80 4-5 1

S 60 40 = so

40 3-5

E 20 30

2-5

100 200 300 400 500 Minutes after suspension Fig. 5. Loss of excess flagella during reversion and retransformation of hairy Naegleria populations. The average flagellum number was determined by making counts of 300 flagellates at 15-min intervals throughout 3 cycles of transformation after the cells were subjected to a 0-45 min 382 °C-temperature shock. Frequency histograms indicate flagellum distribution at times 120, 330 and 480 min post-suspension. O—O, % flagellates; • - - ;f/F.

JjF value greater than the original 25 °C-control suspension. Furthermore, since all clones subjected to the 38 °C-temperature shock developed approximately the same average flagellum number, all cells (clones) in a population must exhibit equal potential for excess flagellum development. Hairiness is therefore a developmental rather than a genetic phenomenon, in the sense that elevated flagellum numbers are not heritable but may be induced in cells undergoing a phenotypic alteration. Because Naegleria flagellates are stable for only about 1 h, and because revertant cells will transform through several successive flagellate cycles (Fulton & Dingle, 1967), the question of the longevity of the excess basal bodies and flagella can be viewed over a much shorter time scale than previously discussed. One can in fact look at the persistence of the basal bodies indirectly by repeatedly assaying f/F in a population of hairy flagellates through a sequence of transformation, reversion, 474 A. D. Dingle retransformation, etc. The results of such a long-term temperature-shock experiment are shown in Fig. 5. The cells transformed normally (Tm = 80 min) to about 96% flagellates by 105 min after suspension. After approximately 60 min these flagellates began reverting to amoebae until, at 240 min, only 30% of the cells were still flagellated. Then the population went through 2 more cycles of transformation and reversion, reaching 95 % and 85 % flagellates, respectively, at 320 and 480 min after suspension. Frequency histograms of flagellum distribution based on counts of 300 cells are included in this figure to illustrate the great excess of flagella on cells following the temperature-shock transformation and the return to normal distributions during the second and third cycles of transformation. Values of f/F were calculated for similar counts made at 20-min intervals throughout the entire 8-h sampling period, and are included in Fig. 5 (closed circles). The maximum value of 4-4 f/F was attained even before the population had become completely flagellated and this value remained essentially constant for approximately 80 min, about 20 min beyond the plateau for percent flagellates. Then f/F dropped rapidly, parallel to the drop in percent flagellated cells, and continuing down to 2-^f/F, i.e. to the normal value. Thus the excess basal bodies and flagella resulting from the heat shock do not survive beyond the initial transformation-reversion cycle. Coulter counts of samples taken at constant threshold values dropped slightly, from 4-2 to 3-2 x io5/ml, indicating some cell loss and/or shrinkage during the 8-h sampling interval, but no cell growth or division. This is significant inasmuch as it rules out the possibility that the drop in f/F could be a simple dilution of organelles accompanying cell division.

DISCUSSION Despite the keen interest presently attracted by studies of flagellar structure and function, little attention has been paid to the differences in flagellum number and distribution encountered in different cells. In fact the question of what cellular controls are exerted over the number and distribution of flagella on different cell types is scarcely posed in the current literature. This interesting and obviously fundamental problem relates primarily to cytodifferentiation in multicellular organisms yet, as is often the case, may be clearly defined and more conveniently studied in one of the so- called 'unicellular differentiating systems'. In this case, the rapid and relatively synchronous transformation of large populations of Naegleria amoebae into biflagellate cells provides the baseline for study. In the transformation under standard conditions (demineralized or glass-distilled water, or dilute tris buffer at 25 °C) there is already evidence of some lability in the control of flagellumnumber . For example, while 65-70 % of the cells in the population have 2 flagella, others commonly develop 3, 4 or (rarely) even higher numbers. It seems reasonable to speculate that 4/(4 flagella) cells are the product of cell fusion or of uneven or incomplete cytokinesis, and the i/and 3/categories could be interpreted as resulting from a mechanical loss of 1 flagellum in cells of the 2/and 4/categories. However, the occasional observation of cells which are of average size yet have up to Control of flagellum number in Naegleria 475 11 and 12 flagellamake s equally plausible the suggestion that some relatively flexible control governs the synthesis and/or assembly of basal bodies and flagella in cells undergoing the amoebo-flagellate transformation. This hypothesis is supported by the fact that experimental interference, in the form of a sublethal temperature shock, consistently induces the formation of excessive numbers of basal bodies and flagella in transforming cells. The increase in basal body and flagellum number is not associated with any cellular growth or fusion phenomenon: it appears to be the result of a developing organelle system escaping from controls which are, at best, somewhat lax. It is interesting to speculate on the nature of replicative events which might result in the observed distribution of flagellum numbers in the hairy populations. Under standard transformation-conditions, the distribution is approximately 3-71-10-15-1, corresponding to 1/, 2/, 3/, etc. If each cell responded to the temperature shock by synthesizing or assembling exactly 2 basal bodies and flagella for each one which would normally develop, the distribution should become 0-3-0-71-0-10-0-15-0-1. The actual distribution of 1-4-8-22-22-13-5-3-1 differs so greatly from the pre- diction that this hypothesis cannot be correct. Even if one transposes all counts from the uneven classes to the next higher even class (on the assumption that uneven counts represent primarily the loss of one flagellum), the inconsistencies between this 'corrected' distribution (0-5-0-30-0-44-0-18-0-4) and the predicted one are still too great to permit acceptance of the simple hypothesis that every basal body normally present replicates once. To be consistent in this approach, one should initially assume that basal bodies or their progenitors are paired in normal populations and that the i/and 3/categories in these counts are artifacts caused by single flagellum losses. The true basal body distribution in normal flagellate populations then ought to read 0-74-0-25-0-1 (by grouping all uneven counts with the next higher even category). A single round of replication affecting all cells in such a population equally would result in a distribution of 0-0-0—74-0-0-0—25—0-0-0-1, which is obviously not the case in real populations. It may not be particuarly profitable to attempt further speculation about the nature of replicative events which might result in the observed patterns of flagellum distribution, for it is obvious that not all cells in the population respond equally to a high- temperature shock. Some cells develop only the 1, 2, 3 or 4 flagella characteristic of 'normal' flagellates. Others develop anywhere from 1 to 14 additional flagella (the maximum recorded on a single cell was 18). Furthermore, if one chooses to interpret the hairiness phenomenon as a process of replication of basal bodies or their progenitors, the simple observation that more than 50% of the cells in a hairy population develop 5 or more flagella implies that some basal bodies or their precursors either replicated more than once or gave rise to multiple progeny. This latter alternative would of course be consistent with patterns of basal body development already established for other diverse sources such as the multiflagellate sperm of Viviparus (Gall, 1961), the blepharoplast-centriole transitions in Marsilea and Zamia (Mikuzami & Gall, 1966) and ciliogenesis in mammalian epithelia (Stockinger & Cirelli, 1965; Dirksen & Crocker, 1966; Sorokin, 1968). However, there is not yet available any morphological or experimental evidence which compels one to interpret centriole 476 A. D. Dingle development as a process of autonomous duplication. Close association of parent and daughter centrioles may reflect only a preferred site of assembly and not necessarily syntheses directed by the nucleic acids such as have been tentatively demonstrated in basal bodies (Seaman, i960; Hoffman, 1965; Randall & Disbrey, 1965; Smith- Sonneborn & Plaut, 1967). Centrioles and basal bodies may instead be derived from syntheses directed by the chromosomes and the supernumerary basal bodies and flagella formed in hairy Naegleria flagellatesma y result from derangements in transcriptional or translational controls at the nuclear gene and ribosome level. Should this be the case, one could not expect to interpret the patterns of flagellum distribution in hairy populations in terms of 'rounds of replication' of the original 1 or 2 basal bodies or their progenitors. The question of the origin of materials and the site of the deranged controls which permit development of excessive numbers of flagella in these cells cannot be answered by the present study. The popular notion that centrioles and centriole-like structures (basal bodies, kinetosomes) arise by division of pre-existing centrioles carries with it the implication of a genetic continuity of centrioles from one generation to the next, and the corollary that cells without centrioles cannot acquire centriole-like structures. Thus, reports of discontinuous inheritance of basal bodies in Naegleria (Schuster, 1963; Dingle & Fulton, 1966) have been questioned, particularly in view of the demonstration that in the water mould Allomyces basal bodies develop from centrioles which though only 160 nm in length and relatively obscure, are present in the vegetative hypha prior to gametogenesis (Renaud & Swift, 1964). In this context it is most interesting that the supernumerary basal bodies and flagellao f hairy flagellates are not inherited by subsequent generations of cells. This is shown by the normal appearance and flagellum number (approximately 2-1 f/F) of 12 separate clones isolated from one temperature-shock experiment and transformed under standard conditions. Amoebae which have reverted from hairy flagellate populations retransform within approximately 60 min. In this short interval, and without any intervening cell division, the flagellum number in the population is reduced from 4-4 to 2-4 fJF. This decrease in fjF begins at the same time as, and continues in parallel with, the reversion to amoebae (recall that fjF is determined only for flagellated cells, so that changes in fjF are independent of the percentage of cells which are flagellated). These observations can be interpreted in various ways. The simplest and most orthodox explanation is that basal bodies are stable and are retained through several rounds of transformation, but that supernumerary basal bodies either degenerate or remain intact but are incapable of organizing flagella. Alternatively, all basal bodies as well as flagella may be lost to the cells upon reversion, necessitating the elaboration of a complete new set of organelles at each subsequent round of transformation. If all basal bodies and flagella were being synthesized anew during subsequent rounds of transformation, it would be reasonable to expect that similar controls would be operative and that a temperature shock equivalent to that which originally induced the hairiness phenomenon should once again result in f/F values approximately double those of normal flagellate populations. This does not appear to be the case, for populations of hairy cells subjected to a second 50- or 60-min temperature shock at various intervals Control of flagellum number in Naegleria 477 during reversion and retransformation develop average flagellum numbers ranging only from 2-13 to 2-58 fjF. The ultimate demonstration of basal body stability or instability in Naegleria awaits the completion of quantitative electron-microscope studies of normal and temperature-shocked cells at various stages of transformation, reversion and retransformation. The present study contributes a method for inducing additional basal bodies and flagella in transforming populations of Naegleria. Given the rapid, synchronous and repetitive transformation (Fulton & Dingle, 1967) methods for producing highly synchronized populations of dividing amoebae (Fulton & Guerrini, 1969), and this temperature-shock method for inducing multiple basal bodies and flagella, it is apparent that the amoebo-flagellate transformation of Naegleria provides a most attractive system with which to further study problems of the continuity and development of basal bodies.

This research was initiated in the laboratory of Dr C. Fulton, Brandeis University, where it was supported by grants from the National Science Foundation and the National Institutes of Health (Institute of Child Health and Human Development). Grants from the National Research Council of Canada supported the completion of this work. I am indebted to Dr Fulton for his continuing interest and stimulating comments on the problem, and to Miss Jane Wills, whose skilful technical assistance is gratefully acknowledged.

REFERENCES ALEXIEFF, A. (1912). Sur les caracteres cytologiques et la systematique des amibes du groupe limax (Naegleria nov. gen. et Iiartmannia nov. gen.) et des amibes parasites des vertebres (Proctamoeba nov. gen.). Bull. Soc. zool. Fr. 37, 55—74. COULTER, H. D. (1967). Rapid and improved methods for embedding biological tissues in Epon 812 and Araldite 502.^. Ultrastruct. Res. 20, 346-355. DINGLE, A. D. (1967). Evidence for a temperature-sensitive control over flagellum development in transforming cells of Naegleria gruberi. J. Protozool. 14 (Suppl.), 22. DINGLE, A. D. & FULTON, C. (1966). Development of the flagellar apparatus of Naegleria. J. Cell Biol. 31, 43-54. DIRKSEN, E. R. & CROCKER, T. T. (1966). Centriole replication in differentiating ciliated cells of mammalian respiratory epithelium. An electron microscopic study. J. Microscopie 5, 629-644. FAWCETT, D. W. (1958). The structure of the mammalian spermatozoan. Int. Rev. Cytol. 7, 195-234- FAWCETT, D. W. (1961). Cilia and flagella. In The Cell, vol. 2 (ed. J. Brachet & A. E. Mirsky), pp. 217-297. New York and London: Academic Press. FULTON, C. (1969). Amebo-flagellates as research partners. In Methods in Cell Physiology, vol. 4 (ed. D. Prescott). New York and London: Academic Press (in the Press). FULTON, C. & DINGLE, A. D. (1967). Appearance of the flagellate phenotype in populations of Naegleria amebae. Devi Biol. 15, 165—191. FULTON, C. & GUERRINI, A. M. (1969). Mitotic synchrony in Naegleria amebae. Expl Cell Res. 56, 194-200. GALL, J. G. (1961). Centriole replication. A study of spermatogenesis in the snail Viviparus. J. biophys. biochem. Cytol. 10, 163-193. HALL, R. P. (1953). Protozoology. Englewood Cliffs: Prentice-Hall, Inc. HOFFMAN, E. j. (1965). The nucleic acids of basal bodies isolated from Tetrahymena pyriformis. J. Cell Biol. 25, 217-228. KERR, N. S. (i960). Flagella formation by myxamoebae of the true slime mold, Didymium nigripes. J. Protozool. 7, 103-108. 478 A. D. Dingle MIZUKAMI, I. & GALL, J. (1966). Centriole replication. II. Sperm formation in the fern, Marsilea, and the cycad, Zamia. J. Cell Biol. 29, 97-111. RANDALL, J. T. & DISBREY, C. (1965). Evidence for the presence of DNA at basal body sites in Tetrahymena pyriformis. Proc. R. Soc. B 162, 473-491. RENAUD, F. L. & SWIFT, H. (1964). The development of basal bodies and flagella in Allomyces arbusculus. J. Cell Biol. 23, 339-354. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy.^. Cell Biol. 17, 208-212. SCHUSTER, F. L. (1963). An electron microscope study of the amoebo-flagellate, Naegleria gruberi (Schardinger). I. The amoeboid and flagellate stages. J. Protozool. 10, 297-312. SEAMAN, G. R. (i960). Large-scale isolation of kinetosomes from the ciliated protozoan Tetra- hymena pyriformis. Expl Cell Res. 21, 292-302. SHELDON, R. W. & PARSONS, T. R. (1967). A Practical Manual on the Use of the Coulter Counter in Marine Science. Toronto, Canada: Coulter Electronic Sales Co. SMITH-SONNEBORN, J. & PLAUT, W. (1967). Evidence for the presence of DNA in the pellicle of Paramecium. J. Cell Sci. 2, 225-234. SOROKIN, S. P. (1968). Reconstruction of centriole formation and ciliogenesis in mammalian lungs. J. Cell Sci. 3, 207-230. STOCKINGER, L. & CIRELLI, E. (1965). Eine bisher unbekannte Art dcr Zentriolenvermehrung. Z. Zellforsch. mikrosk. Anat. 68, 733-740. WHEATLEY, D. N. (1967). Cells with two cilia in the rat adenohypophysis. jf. Anat. 101, 479-485- WILLMER, E. N. (1956). Factors which influence the acquisition of flagella by the amoeba, Naegleria gruberi. J. exp. Biol. 33, 583-603. WILSON, C. W. (1916). On the life-history of a soil amoeba. Univ. Calif. Publs Zool. 16, 241-292.

{Received 14 January 1970) Control of flagellum number in Naegleria 479

Fig. 6. Phase-contrast photomicrograph of a field of 'normal' flagellates fixed after 120 min transformation at 25 °C. x 600. Inset. Typical biflagellate cell from the same sample, x 2500. Fig. 7. Phase-contrast photomicrograph of a field of temperature-shocked flagellates fixed in Lugol's iodine 120 minutes after suspension, x 600. Inset. A single hairy flagellate from the same sample with 8 flagellavisible , x 2500. 480 A. D. Dingle

Fig. 8. Electron micrograph of a portion of a hairy flagellate illustrating 4 basal bodies, 2 of which are sectioned longitudinally and 2 transversely. Note that a rhizoplast extends from each pair of basal bodies. A bundle of cytoplasmic microtubules under- lies the cell membrane, running at right angles to the long axis of the basal body (bb"). (bb1'2, longitudinally sectioned basal bodies; 6i3'4, transversely sectioned basal bodies; int, cytoplasmic microtubules; n, nucleus; r1'2, rhi'soplasts.) x 28 000. Control of flagellum number in Naegleria 481

Fig. 9. A cluster of 5 basal bodies in the cytoplasm of a hairy flagellate, illustrating the typical centriole-like cartwheel structure of the basal bodies, and the series of filaments which seem to connect the basal bodies to the splayed branches of the periodically banded rhizoplast. x 80000. Fig. 10. All transversely sectioned flagella observed in populations of hairy flagellates of Naegleria have the typical 9 + 2 structure shown in this figure, x 120000.