Studies on the Rhizoplast from Naegleria Gruberi

J. Cell Sci. 47, 277-293 (1981) 277 Printed in Great Britain © Company of Biologists Limited IQSI

STUDIES ON THE RHIZOPLAST FROM NAEGLERIA GRUBERI

PETER R. GARDINER*, ROBERT H. MILLER AND MARK C. P. MARSHf Department of Zoology, University College London, Gotver Street, London WCiE 6BT, England

SUMMARY A procedure, utilizing homogenization and centrifugation in a low ionic strength buffer containing Triton X-100, has been used to facilitate the isolation of the rhizoplast from flagellates of Naegleria gruberi. This has enabled a study to be made of the physical and biochemical properties of this organelle. The rhizoplast is shown to be a proteinaceous structure with chemical properties similar to those of the molluscan gill ciliary rootlet. Polyacrylamide gel electrophoresis gives a possible subunit molecular weight of approximately 240000 Daltons. Studies with antisera raised against the rhizoplast fraction demonstrated the absence of rhizoplast antigens in amoeboid forms of Naegleria gruberi and is taken as evidence that the organelle is synthesized de novo during transformation of the amoeba to the flagellate form. Results of optical diffraction studies on isolated rhizoplasts are also presented.

INTRODUCTION The striated flagellar rootlet, the rhizoplast, of the flagellate phenotype of the protozoon Naegleria gruberi offers an unusually accessible example of organelle development in an eukaryote cell. The rhizoplast appears during the amoeba-to- flagellate transformation and is subsequently broken down on reversion of the temporary flagellatestag e to the amoeboid form. A ribonucleic acid (RNA) and protein synthesis requirement for amoeba-to-flagellate transformation was shown by actino- mycin D and cycloheximide sensitivity (Walsh & Fulton, 1973; Preston & O'Dell, 1973, 1974) and recently by in vitro translation of transforming cell specific mRNA (Lai, Walsh, Wardell & Fulton, 1979). Neither process is required for the flagellate- to-amoeba reversion. With Naegleria gruberi it is possible to induce phenotypic changes synchronously in populations of cells by simple laboratory manipulation (Preston & O'Dell, 1973). We have attempted to use the rhizoplast system to investigate the molecular mechanisms regulating gene expression and organellogenesis in Naegleria. Here we describe biochemical and structural studies on isolated rhizoplasts and present evidence indicating the rhizoplast to be an organelle specific to the flagellate phenotype. The results suggest that the rhizoplast component(s) are synthesized de novo during

• Present address: National Institute of Allergy and Disease, National Institute of Health, Bethesda, Maryland, U.S.A. f Address for correspondence: The European Molecular Biology Laboratory, Postfach 10.2209, 6900 Heidelberg, F.D.R. 278 P. R. Gardiner, R. H. Miller and M. C. P. Marsh transformation and offer a second example of synthesis and assembly of components of the mastigont apparatus in Naegleria gruberi, after the demonstration that at least 70% of the flagella outer-doublet tubulin (Kowit & Fulton, 1974a, b) and 92% of the flagellar tubulin mRNA (Lai et al. 1979) are synthesized de novo during the amoeba- to-flagellate transformation.

MATERIALS AND METHODS Cell culture and transformation Naegleria gruberi (Cambridge Culture Collection Stock 1518/1C) was maintained in monox- enic culture with Escherichia coli on o-2 % (w/v) peptone agar plates. Bulk culture methods were employed to give the large number of cells (> io8) required for rhizoplast isolation. Flat-bottomed 530-011' Pyrex dishes were layered with 200 ml nutrient agar. E. coli from a confluent 9-cm diameter bacterial plate, and Naegleria gruberi cysts from 5x9 cm stock plates were seeded onto 5 Pyrex dishes. The dishes were flooded with a few millimetres of sterile distilled water to encourage excystation and to spread the bacteria and protozoa. The dishes, covered with aluminium foil, were kept for 3-4 days at 27 °C. Amoebae were harvested by scraping the agar surfaces with a glass rod. Large agar debris was removed by pouring the amoeba suspension through a Swinney filter support screen (Millipore Ltd.). The cells were pelleted by centrifugation at 750 g for 20 min at 4 °C, and washed in 200 ml of 2 mM Tris-HCl buffer, pH 7-6. The cells were repelleted (550 g for 15 min), resuspended in 3 ml of the same buffer and layered onto a discontinuous Ficoll (Sigma) gradient, consisting of 4 ml of 20% w/v Ficoll, 15 ml of 10%, 5 ml each of 7-5 % and 5 % in 2 mM Tris-HCl, pH 76. Centrifugation (550 g for 15 min at 4 °C) banded the cells at the 10-20% interface, while bacteria and fine agar remained in the 10% layer. Remaining Ficoll was removed by washing and the cells were counted in a model ZBi Coulter Counter. Five large dishes usually yielded 1-2 x 10' cells, though larger harvests could be obtained after incubation of the dishes for up to 5 days, but with an increased cyst-to-cell ratio. The amoeba-to-flagellate transformation was synchronized by resuspending the cells in 66 % (v/v) D,O (Preston & O'Dell, 1973). After 45 min the cells were centrifuged (550 g for 15 min at 4 °C) and the pellet resuspended at 5 x io5 cells ml"1 in 2 mM Tris-HCl, pH 7-6 on a shaking 30 °C waterbath. After 120 min aliquots were fixed with Lugol's iodine and the percentage of flagella bearing cells determined under phase-contrast or Nomarski microscopy. Large cyst loads lowered the percentage of transformed cells; however, between 60 and 70% flagellates was considered sufficient for experimental purposes.

Isolation of rhizoplasts Flagellates, pelleted at 550 g for 10 min at 4 °C, were immediately resuspended in 3-5 ml of 30 mM Tris-HCl, pH 80, containing 05 % Triton X-100 and 3 mM MgClj in a 2-cm diameter glass homogenizer fitted with a teflon pestle (Tri-R Instruments, Camlab.) on ice. The pestle was motor-driven at 500 rev/min and 75 passages of the pestle in the tube were made during a 3-min homogenization. Homogenization disrupted all motile phenotypes and broke most of the cysts. The homogenate was centrifuged at 180 g for 10 min at 4 °C to sediment cysts, cyst cases and large cell debris. The supernatant was centrifuged at 950 g for 20 min to produce a second pellet, P,, which light and electron microscopy showed to contain intact rhizoplasts. P, was the source of the rhizoplasts in subsequent experiments. The P. fraction was further treated with ribonuclease B and lipase (2 mg ml-1) at 37 °C for 30 min to remove ribonuclear or lipid material. This was subsequently modified to a 30- min treatment with ribonuclease A and B (0-5 mg ml"1), deoxyribonuclease (0-15 mg ml"1) and phospholipase C (0-5 mg ml"1) at 37 °C (all enzymes from Sigma). Enzyme action was stopped by dilution of the sample into 15 ml of 2 mM Tris-HCl, pH 7-6, at o °C, and the rhizoplasts were pelleted by centrifugation (950 g for 20 min at 4 °C). Studies on the rhizoplast from Naegleria gruberi 279

Transmission electron microscopy Whole cells and cell fractions were fixed for 30 min at room temperature or overnight at 4 °C with 15-20% (v/v) glutaraldehyde (TAAB laboratories). After washing, the samples were postfixed in 1 % OsCv, for 60 min, agar embedded, dehydrated in ethanol-water mix- tures and embedded in Araldite. Silver-gold sections were stained with ethanolic uranyl acetate and lead citrate.

Negative staining Five-microlitre samples of P, fractions before or after treatment with reagents, were dried onto carbon-coated copper grids. The samples were fixed with unbuffered 2'5 % (v/v) glutaraldehyde, stained with 2 % aqueous uranyl acetate and the grids dried on filter paper. Specimens were examined in an AEI-EM6B electron microscope operated at 60 kV.

STEM and scanning electron microscopy Negatively stained rhizoplasts, without further treatment, or rhizoplasts which were critical- point dried and briefly shadowed with platinum, were viewed by the scanning-transmission mode (STEM) or surface-scanning mode in a JEOL 100 CX TEMSCAN electron microscope at accelerating voltages of 40 or 100 kV.

Measurement of the rhizoplast's dimensions A Wild phase-contrast microscope with x 50 oil-immersion objective (N.A. i-o) and a drawing tube was used to trace the outlines of 100 rhizoplasts onto graph paper. A measuring wheel, calibrated from a micrometer slide, was used to measure the lengths. The periodic banding was measured directly from photographic prints of negatively- stained material enlarged between 125 K and 200 K diameters and by microdensitometric (Joyce-Loebl) scanning of electron micrograph negatives. The negatives of electron micrographs of isolated, negatively-stained rhizoplasts were also analysed by optical diffraction. Negatives of electron micrographs of catalase crystals (Polaron) were used as standards.

Biochemical treatments of the rhizoplast fraction Enzymic and biochemical treatments were carried out either in test tubes, taking sample aliquots at specific times and preparing them for negative staining, or directly on the EM grids. The reactions were terminated by addition of cold 25 % glutaraldehyde. Enzymes and chemicals were supplied as follows: trypsin, collagenase, lysosyme, vinblastine sulphate, EGTA (ethylene glycol-&i'j-(/?-amino ethyl ether) N,N'-tetra-acetic acid) from Sigma; pronase and colcemid from Calbiochem; amyloglucosidase, urea, dithiothreitol, sodium dodecyl sulphate, sodium deoxycholate and deuterium oxide from B.D.H. All agents were used in 30 min Tris-HCl, pH 8-0, unless otherwise stated. Freezing and thawing, using liquid nitrogen, and sonication (for 3 min at 40 W on a Bran- son Sonic Power Co. Sonifier B12, equipped with a microtip) were also carried out.

Electrophoretic analysis Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis was carried out on a discontinuous slab gel system (Laemmli & Favre, 1973). Samples in o-i ml of 0025 M Tris- HCl, pH 6-7, containing 2% SDS, o-i % bromophenol blue, 40% sucrose and 15% mer- captoethanol were heated to 100 °C for 5 min. After i-min centrifugation in a microfuge, 50-/4I samples were loaded onto the gel. The gels were stained with Coomasie brilliant blue, and destained in acetic acid. 280 P. R. Gardiner, R. H. Miller and M. C. P. Marsh

Antisera A P, fraction from 6-2 x io1 flagellates in Freund's complete adjuvant was injected intramuscularly into a rabbit at 6 sites. A second Ps fraction from 7x10' flagellates was injected intramuscularly 3 weeks later, and after a further 3 weeks the rabbit was finally boosted with a P, fraction from i-6 x io8 flagellates. The rabbit was bled 2 weeks later and gamma globulin fraction prepared by ammonium sulphate precipitation.

Immunofluorescent microscopy Pj samples were incubated with 10-fil aliquots of sera or gamma globulin for 20 min at room temperature. After washing with 3 x 3 ml of 015 M NaCl, the pellets were resuspended in 0-2 ml of 015 M NaCl containing 10 /tl of FITC-goat anti-rabbit IgG. After 20 min incubation, the samples were washed as above, and finally with 1 M NaCl to remove non-specific fluorescence. Samples were viewed with a Leitz Orthoplan fluorescence microscope. Antisera were adsorbed with glutaraldehyde-cross-linked immunoadsorbents prepared from homogenates of either flagellateso r amoebae (Avrameas & Ternyck, 1969). Bovine serum albumin (BSA 20 % w/v) was used to raise the protein concentration and enhance the cross- linking. Glutaraldehyde to a final concentration of 25 % was added to a minimum workable volume of the homogenate plus BSA. Cross-linking occurred in approximately 30 min at room temperature and was completed overnight at 4 °C. The gels, broken into fine particles using a Silversen mixer-emulsifier, were washed with phosphate-buffered saline (PBS) until the O.D. of the washings was zero at 280 nm. Protein estimates (Lowry, Rosebrough, Farr & Randall, 1951) indicated at least 95 % of the protein crosslinked. Unreacted aldehyde groups were blocked by washing with 1 % lysine in PBS. The gels were packed into columns (void volume approx. 1 ml) and aliquots (0-25 ml) of sera were run into the column. After incubation for 60 min at room temperature, the non- adsorbed serum was eluted with PBS. Adsorption was assayed initially by cell surface immunofluorescence on formaldehyde-fixed cells and by double-diffusion Ouchterlony precipitation.

RESULTS In situ rhizoplasts We observed the course of the rhizoplast in flagellate forms of N. gruberi utilizing electron microscopy and the lysis technique of Dingle & Fulton (1966). In most cells the single rhizoplast connected the 2 basal bodies to the nucleus, apparently adhering to the nuclear membrane and terminating in the vicinity of the nucleus (Fig. 1). In a few cells the rhizoplast ran alongside the nucleus and continued towards the posterior membrane of the cell (Fig. 2). The mean length of the rhizoplasts was 6-68 /im, but showed wide variation, which was not due to breakage during isolation. Intact rhizoplasts have a characteristic 'elongated tadpole' appearance (resulting from maintained association with the basal bodies) not observed with obviously damaged samples obtained after sonication. Anteriorly the rhizoplast contacts the 2 basal bodies, at the level of the cartwheel, via wedge-shaped filamentous arrays which were observed both in situ and with isolated rhizoplasts (Fig. 3A, B). This interaction showed great tenacity: rhizoplasts and basal bodies could not be separated by chemical or physical means, while flagellar axonemes were easily sheared from the basal bodies. The similarity in the banding pattern of the connecting structures and rhizoplast, combined with the observation Studies on the rhizoplast from naegleria gruberi 281

Fig. 1. Electron micrograph of the anterior of a flagellate showing the usual course of the rhizoplast (arrowheads) from 2 anteriorly placed basal bodies (bb), sectioned at the level of the cartwheel, to the nucleus (n) alongside which the rhizoplast runs for some of its length. Bar, 1 fim. Fig. 2. Montage of a flagellateillustratin g a case where the rhizoplast apparently runs towards the plasmalemma at the posterior of the cell. A straight line can be drawn from the rhizoplast profile, along the side of the nucleus to the anteriorly placed basal bodies. Bar, 1 fim. 282 P. R. Gardiner, R. H. Miller and M. C. P. Marsh Studies on the rhizoplast from Naegleria gruberi 283 that they have comparable sensitivities to chemical treatments, implied that they may share common structural elements. Microtubules and smooth endoplasmic reticulum which have been observed associated with the basal apparatus in Chlamy- domonas (Ringo, 1967; Katz & McLean, 1979) and Polytomella (Brown, Massalski & Patenaude, 1976) were not observed in N. gruberi. The nature of the posterior rhizoplast interaction was less well defined. Many sections of flagellates showed the rhizoplast profiles running towards the nucleus and occasionally towards the posterior membrane of the cell. Physical continuity was not observed and although the nuclear membrane and rhizoplast may come into close proximity, nuclear membrane or material was not observed associated with isolated rhizoplasts.

Isolated rhizoplasts The isolation procedure for the rhizoplast was adapted from that of Simpson & Dingle (1971). The incorporation of Triton X-100 into the homogenization buffer gave a 100-fold improvement in the yield of isolated organelles which, although still not high, gave a reliable source of intact rhizoplasts and enabled biochemical studies to be carried out. Thin sections of a P2 fraction indicate that it is heterogeneous, with membrane and subcellular organelle profiles present (Fig. 4). In samples of P2 after negative staining, the rhizoplasts were easily seen and identified by the elongated-tadpole appearance and by the characteristic banding pattern (Fig. 5). Three different methods gave a major light—dark repeat of 22 nm over the entire length of the organelle (Table 1). In addition, we observed a minor light band occur- ring within the major dark period (Figs. 6, 7). This was observed as a small shoulder on microdensitometer traces of the micrographs. We did not observe any change in the rhizoplast banding periodicity when buffering conditions were changed, or when the calcium concentration was increased or decreased. To obtain further physical measurements of the band-repeat distances of the rhizoplast and to gain some insight into the ordering of the protofilaments within the structure, optical diffraction studies were carried out. Figs. 6 B and 7B are the optical diffraction patterns obtained from the micrograph negatives of the illustrated rhizoplasts (Figs. 6A and 7A respectively). Both diffraction patterns clearly show the major repeat distance of the rhizoplast (22-2 nm), giving up to sixth order diffraction in the equatorial direction. In some transforms the fourth order spot is reduced while the fifth appears slightly stressed. The significance of this is at present unknown.

Fig. 3. Micrographs showing the wedge-shaped, fibrous, basal body connectives, A, in situ (see also Fig. 1) and B, in an isolated and sectioned complex. Bar, 1 fim. Fig. 4. Section through a P, pellet showing rhizoplast profiles (arrowheads) and other inclusions of P, such as axonemal components. Bar, 1 fim. Fig. 5. Whole rhizoplast with basal body complex (bb) from a P, sample fixed on a carbon coated grid and negatively stained. Bar, 1 fim. P. R. Gardiner, R. H. Miller and M. C. P. Marsh Studies on the rhizoplast from Naegleria gruberi 285

Table 1. Dimensions of isolated rhizoplast

Rhizoplast dimension Size Method of determination Mean length 6-68 ± 3•14 fim Profile measurement, phase Range 18' i-2-o /tm microscopy Mean diameter 134 nm Measurement from micrographs Band repeat distance 225 nm Measurement from micrographs 22-o nm Measurement from densitometry 22-2 nm Optical diffraction Subdivision of band repeat Major light band and minor light band! 17-5 nm Measurement from micrographs Thus, dark band between minor and 50 nm next major light band Protofibril diameter 3-0 nm Measurement from micrographs Protofibril centre-to-centre spacing 5-25 nm Optical diffraction Length of region in contact with basal 0-4 «m Measurement from micrographs bodies (in situ)

In Fig. JB the longitudinal filament centre-to-centre spacing (5-25 nm) is illustrated by the single dashes in the meridional direction. To study whether the periodic banding pattern of the rhizoplast is reflected in the surface topography of the organelle, STEM and surface scanning views of isolated organelles were made. However, no evidence for a 'corrugated' structure correspond- ing with the banding pattern observed by negative staining was observed (Fig. 8).

Fig. 6. A. Optical diffraction pattern obtained from the photographic negative of the isolated and negatively stained rhizoplast shown in B. The central halo and slightly off-vertical line are due to the mask around the negative when the pattern was made. The prominent dashes in the near horizontal (equatorial) dimension are interference maxima representing the banded repeat distance of the rhizoplast. Six orders (and their reflexions on the opposite side of the zero line) can be seen. The first-order diffraction spot falls within the central halo. The fourth seems somewhat diminished compared with the rest. B. This isolated and negatively stained rhizoplast had been treated after isolation with 001 % SDS for 10 min and clearly shows the major light band and the dark interband components of the rhizoplast repeat. The interband region is traversed by the previously unreported minor band. Bar, o-i fim. Fig. 7. A. Optical diffraction pattern obtained from the negative of the isolated and negatively stained rhizoplast shown in B. The transform shows the same 6 diffraction orders in the equatorial dimension (representing the band repeat distance) as Fig. 7 A. The first is obscured by the bright central halo whilst the fourth is again noticeably diminished. A single spot in the vertical (or meridional) dimension and its reflexion below the equator represent the centre-to-centre spacing of the longitudinal protofibrils. Above the equator this spot might be composed of 2 parts although this does not show in the somewhat extended dash of the reflexion. The estimate for the centre- to-centre spacing of the protofibril (Table 1) is therefore an average of 3 measurements taken to the nearest, middle and furthest point of the dashes from the centre of the pattern. B. Isolated and negatively stained rhizoplast which had been subjected to o-oi % SDS for 10 min. The major light band and dark interband regions are evident and the light minor bands are prominent. Bar, o-i /im. P. R. Gardiner, R. H. Miller and M. C. P. Marsh

Fig. 8. Scanning and TEMSCAN electronmicrographs of isolated rhizoplasts. A. TEMSCAN view of a rhizoplast clearly showing the periodic banding pattern of the organelle. (Taken at accelerating voltage of ioo kV.) B. Surface scanning view of A; the banding pattern is faintly visible, but was thought to be due to the high accelerating voltages used. Specimen unshadowed, c. Critical-point dried rhizoplast shadowed with platinum, indicating that the rhizoplast appears uncorrugated, but cylindrical in form. Studies on the rhizoplast from Naegleria gruberi 287

Biochemical analysis The tenacity of the rhizoplast-basal body connexions prevented further purification of the organelle. The results of chemical and physical treatments designed to disso- ciate this interaction, and their effect on the rhizoplast itself, are given in Tables 2 and 3. No single method was found which removed selectively the basal bodies whilst leaving the rhizoplast intact (with the possible exception of high pH where a few isolated rhizoplasts were found without basal bodies). The rhizoplast was stable in 10% Triton X-100, and disulphide bond-breaking agents. High concentrations of

Table 2. Effect of enzymes on the integrity of isolated rhizoplasts

Sensitivity Resistance of rhizoplast of rhizoplast Enyzme Activity/ml to treatment, to treatment, min min Trypsin 11400 < 2-5 • 57°°° < 2-5 Pronase 45 < 2-5 2250 < 2-5 Collagenase 400 < 3° 125-4 < 3° . Amyloglucosidase 300 > 3° Lysozyme 40500 > 3° Ribonuclease A 78 > 60 Ribonuclease B 100 > 60 Deoxyribonuclease 7i4 > 60 Phospholipase C 6 > 60 Lipase 1700 > 3°

Enzymes were in 30 mM Tris-HCl pH 8-o containing 3 mM MgCl, and 2 mM CaCla at room temperature. microtubule depolymerizing agents, vinblastine and colcemid, disrupted neither the rhizoplast nor the basal bodies and we presumed that non-tubulin proteins in the complex stabilized the basal bodies. The rhizoplast was sensitive to extremes of pH, but not to high ionic strength for periods in excess of 5 min. The rhizoplast was disrupted by slow freezing and thawing, but was stable when kept at 4 °C overnight or when passed through 5 cycles of 'snap freezing' and thawing, with no disruption of the basal bodies. Sonication broke the rhizoplast into fragments, along presumed transverse planes of weakness, without concomitant loss of the periodic banding pattern within the fragments. Some fragments retained the basal body and were presumed to be the most proximal pieces of disrupted organelles, but more distal fragments without basal bodies co-sedimented with these fragments on centrifugation.

Electrophoresis

SDS-PAGE analysis of P2 indicated a protein band with an apparent mol. wt in the range of 220-240 kD, which constituted the major protein component of Pa 288 P. R. Gardiner, R. H. Miller and M. C. P. Marsh (Fig. 9). Some evidence that this band was a rhizoplast component came from experiments in which chemically treated P2 samples were analysed. In samples treated with EDTA and EGTA, conditions which do not solubilize the rhizoplast, the 240 kD band remained associated with the pelleted material (30 min at 950 g). In P2 samples treated with 0-5 % SDS (which rapidly solubilized the rhizoplasts) the 240 kD band was found in the supernatant. In collagenase-treated samples, where the rhizoplast was disrupted, there was a decrease or complete removal of the high-molecular- weight band from treated samples. Amoebae samples prepared in the same way as

Table 3. Effects of different reagents on the integrity of isolated rhizoplasts

Sensitivity, Resistance, Reagent min time

Triton X-ioo 10% 24 h Deoxycholate < i-o SDS, % o-os < 025 O-O2O < i-o > 10 min Urea 8 M < 0-25 Dithiothreitol 20 mM >• 5 min Colcemid, 50 mM o °C (in DMSO 50 %) > 24 h Vinblastine 10 mM > 24 h Calcium (CaCl,), 10 mM o °C > 24 h EGTA 30 mM \ No Mg1 + 30 h EDTA 10 mM/ in buffer {I 30 h D,O ioo%o°C . > 16 h Glycine buffer, 10 mM pH 3-0 < 2-O 31 ± ± 3-2-3-5 > 30 h Universal buffer pH 7-6-9-4 > 30 h 95 < 30 96 < 5 NaCl, 2 M > 30 h Agents, unless otherwise stated, were in 30 mM Tris-HCl pH 8-0 containing 3 mM MgCl, at room temperature.

P2, or whole amoebae extracts, do not contain a similar high molecular weight band. Further purification is required to demonstrate unequivocally that this band is a rhizoplast component.

Immunofluorescent staining of the rhizoplast

A double-layer indirect immunofluorescence technique was used (Table 4). Pa fractions were incubated with antisera and a second label of FITC-goat anti-rabbit IgG. The anti-P2 serum contained activity against components of the amoeba and flagellate cell surface and activity which stained the rhizoplast. The activity against the rhizoplast was removed by adsorption of the anti-P2 sera with a flagellate-immunoadsorbent, but not by an amoeba-immunoadsorbent. Removal of activity from Studies on the rhizoplast from Naegleria gruberi

a b c d e

Fig. 9. SDS-polyacrylamide gel electrophoresis patterns of rhizoplast samples. Proposed rhizoplast bands indicated by arrows, A. Gel samples were: a, RNA poly- merase; b, BSA and trypsin inhibitor; c, Ft pellet; d, P, pellet after 30 min treatment with collagenase, demonstrating reduced high-molecular-weight band; e, actin. B. P, pellet samples, showing the prominent high-molecular-weight band. the flagellate-adsorbed anti-P2 was not due to dilution, as indicated by the continued presence of the activity after passage through amoeba columns, and by control dilution experiments. The cell surface activity was removed by passage through both flagellate and amoeba-immunoadsorbents.

DISCUSSION Striated fibres are found associated with the mastigont apparatus of a variety of ciliated and flagellated cells (Pitelka, 1969; Eyden, 1976). Generally they are considered to anchor the flagellar bases. However, in cases where they connect basal 290 P. R. Gardiner, R. H. Miller and M. C. P. Marsh bodies, more complex functions have been ascribed, e.g. to coordinate flagellar beat (Ringo, 1967; Hyams & Borisy, 1975), or to transmit stimuli between the basal apparatus and the mating structure in Chlamydomonas (Goodenough & Weiss, 1978). However, in Naegleria we believe that the association of the rhizoplast with the nucleus, and also possibly with mitochondria (observed aligned along the rhizoplast) effectively increases the bulk of the organelle which, together with the rhizoplast's resistance to flexion and the viscosity of the flagellate's cytoplasm, serves to restrict the oscillations of the basal bodies during flagellar motion. Three methods give a major light-dark periodicity between 22-0 and 22-5 nm for the isolated rhizoplasts. The most precise, optical diffraction, gives a constant value of 22-2 nm for 4 samples from which optical transforms were obtained. However, a large variation can occur in the periodicity of rhizoplasts in situ (between 12 and 26 nm). Such a variation was also observed by Simpson & Dingle (1971), who suggested that the rhizoplast had elastic or contractile properties, and that isolated rhizoplasts

Table 4. Immunofluorescent staining of the rhizoplast

Cell surface Immunoadsorbent Rhizoplast fluorescence Serum used fluorescence (Amoeba) Rabbit IgG Anti-amoeba • • + + + Anti-amoeba Amoeba Anti-P, . + + + + + + Anti-P, (1:8 dil.) . + + + + + + Anti-P, Amoeba + + + Anti-P, Flagellate Anti-P, BSA + + + + + + showing little variation in periodicity could be considered relaxed. Whether the organelle is actively contractile or whether the variable periodicity is a result of tensile or compressive forces exerted on the organelle has not, as yet, been resolved. An observed variation of the periodicity of different in situ rhizoplasts within the same section suggests that the variation is not due to preparative artefacts. However, an observed variation in the periodicity of isolated organelles prepared for transmission electron microscopy (the negatively stained specimens retain a 22-nm period) suggests that the difference may arise from differences in the methods of preparation. The periodicity of the striated flagella rootlet of Platymonas subcordiformis is sensitive to changes in the free calcium concentration, the organelle undergoing a calcium- induced contraction (Salisbury & Floyd, 1978). We have not observed such changes with the Naegleria rhizoplast at free calcium concentrations up to 10 mM, or in the presence of calcium chelators. In addition, Katz & McLean (1979) suggest that the smooth endoplasmic reticulum associated with the basal complex of C. tnoewusii may act as a calcium sink for calcium-induced contractile rootlets. Such elements are not associated with the Naegleria rhizoplast/basal body complex. Sleigh (1979) com- mented on the possible implication of contractile striated organelles in flagellate and Studies on the rhizoplast from Naegleria gruberi 291 ciliary systems, but behavioural studies on Naegleria flagellates have not been carried out and information on the coordination of the flagellar beat is not available. A calcium- induced contractile system would perhaps be unlikely in Naegleria as the flagellates are highly sensitive to free calcium and undergo rapid reversion to the amoeba in the presence of 1 mM CaCl2 (Fulton, 1977a; O'Dell & Blair, 1977). The dashes in the meridional dimension of the optical transforms, which reflect the centre-to-centre spacing of the longitudinal filaments of the rhizoplasts, result from improper alignment of the protofibrils. This may suggest an orthogonal dis- placement of the filament9 from the long axis of the rhizoplast caused by overlap in the region of the band. The centre-to-centre spacing of 5-25 nm represents the maxi- mum possible diameter for the protofibrils and is comparable with the 5-nm subfibrils of ciliate kinetodesmal fibres (Rubin & Cunningham, 1973) and the 4-5 nm subfibrils of the molluscan gill ciliary rootlet (Stephens, 1975). The rhizoplast of N. gruberi appears to be composed of longitudinal protofilaments arranged in parallel. The light—dark banding pattern and the somewhat corrugated edge of negatively stained rhizoplasts (Figs. 6, 7) suggest, by analogy to isolated fibres of paramyosin, that the light bands are regions of molecular overlap excluding the stain, whilst the dark bands, where stain has penetrated, are regions of lower protein concentration (Cohen, Szent-Gyorgyi & Kendrick-Jones, 1971). The asym- metry of the minor band, always to one side of the major light band, suggests that the banding pattern arises from a polar array of molecules (an anti-parallel array is required for a symmetric pattern: Kend.ick-Jones, Szent-Gyorgyi & Cohen, 1971; Weisel & Szent-Gyorgyi, 1975). However, without knowing whether the minor band represents a protuberance of the protofibril or a cross-link, rather than a true region of overlap, further speculation on the molecular organization of the organelle is not possible. The rhizoplast is sensitive to proteases, charged detergents, urea and extremes of pH, but not to microtubule-disrupting agents and disulphide-bond breaking agents. The rhizoplast differs, in its detergent sensitivity, from the molluscan gill ciliary rootlet which is resistant to 1 % hot SDS (Stephens, 1975), but both organelles are soluble at low pH. The high and low disrupting pH, and the sensitivity to chaotropic agents suggests that hydrogen bonding is required for the integrity of the organelle. The maintenance or disintegration of the rhizoplast was paralleled in all cases by that of the basal bodies which may reflect an interdependence for structural stability between the 2 organelles.

A 240 kD protein constitutes the major protein species of the P2 fraction (the band does not migrate with rabbit skeletal muscle myosin, which in our system runs at 200 kD). A rhizoplast component protein of this molecular weight would be com- patible with other striated rootlet systems for which biochemical data are available, for example, the molluscan gill ciliary rootlet has 230 and 250 kD components (Stephens, 1975) and the kinetodesmal fibre protein from 3 strains of Tetrahymena has a mol. wt of 250 kD (Vaudaux, Williams, Frankel & Vaudaux, 1977). Dingle & Green (1974) reported that the rhizoplast of N. gruberi migrated as a single protein species, on SDS-PAGE, with a mol. wt of 240 kD. However, Fulton (1977b) reports 292 P. R. Gardiner, R. H. Miller and M. C. P. Marsh that Larson & Dingle found the rhizoplast to be composed of a single 160-kD protein. The rhizoplast does not, however, appear to have any biochemical homology with the striated costa of Trichomonas (Amos, Grimstone, Rothschild & Allen, 1979). Pre- paration of an antiserum against the 240-kD band should enable us to clarify whether or not this band is derived from the rhizoplast. We found no evidence to support suggestions that the rhizoplast is composed of a primitive form of collagen (Schuster, 1963; Dingle & Green, 1974). Collagen, in all cases to date, is an extracellular protein and there is only one report of a collagen-like protein in the protozoa (Haliphysena), where it is also extracellular (Hedley & Wake- field, 1967). The molluscan gill ciliary rootlet (Stephens, 1975) and the rhizoplast are highly sensitive to trypsin but collagen is resistant. Native collagen is composed of a and /? 100-kD subunits and collagen stains pink with Coomassie blue (Stephens, 1975) due to the high hydroxyproline content. No pink-staining bands were observed in our system. Collagenase does cause slow dissolution of the rhizoplast, but these enzymes from bacterial sources can have broad specificities (Mihalyi, 1972). The results from immunofluorescence studies indicate that the anti-P2 antiserum contained anti-rhizoplast activity, which could not be removed by preincubation with an amoeba immunoadsorbent but could by a flagellate immunoadsorbent. This implies that either the rhizoplast components are not present in the amoeba prior to transformation or that they exist in a different antigenic form. However, the failure to detect a 240-kD band in extracts of amoebae run on SDS-PAGE suggest that the former alternative is more likely. The rhizoplast may present a second example of de novo synthesis of specific components during the amoeba-to-flagellate transformation in N. gruberi. Further studies and improved purification methods should allow a fuller exploitation of this system. We would like to thank Mr David Holberton for his assistance and advice with the optical diffraction studies, Dr Susan Cotmore for critically reading the manuscript, Andrew Wiffen for technical assistance and Wendy Moses for typing the manuscript.

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