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A short flagella mutant of Dunaliella salina (Chlorophyta, Chlorophyceae)
Micron 35 (2004) 337–344 www.elsevier.com/locate/micron
A short flagella mutant of Dunaliella salina (Chlorophyta, Chlorophyceae) Rosa Vismaraa, Franco Vernib, Laura Barsantia, Valtere Evangelistaa, Paolo Gualtieria,* a Istituto di Biofisica CNR, Area della Ricerca Pisa, Via Moruzzi 1, 56124 Pisa, Italy Dipartimento di Etologia, Ecologia, Evoluzione, Universita` degli Studi di Pisa, Via A. Volta 6, 56127 Pisa, Italy
b
Abstract Dunaliella salina (Chlorophyta, Chlorophyceae) is a unicellular wall-less biflagellate alga. In this paper we describe a spontaneous mutant of D. salina, isolated from wild type cultures, which is characterized by very short flagella. The ultrastructure showed the basic 9 þ 2 organization of wild-type flagella. Immunofluorescence localization of tubulin in this mutant confirmed the normal construction of the axoneme. Although, the mutant does not swim, still it is able to move and perform photobehavior. As shown by track reconstruction, and rotation movements, observed by means of reflection microscopy, this mutant can move, probably gliding by means of its stumpy flagella. A possible model to explain the mutant motion pattern is discussed. q 2004 Elsevier Ltd. All rights reserved. Keywords: Mutant; Photobehaviour; Track reconstruction; Flagella; Gliding
1. Introduction The mechanisms that determine and preserve the size and function of cellular organelles represent a fundamental question in cell biology, which up to now has been only partially understood. Cilia and flagella, in metazoan as well as in unicellular organisms, provide a handy model system to investigate size-control analysis of organelles. The phenomenon of intraflagellar transport (IFT) provides evidence that flagella are dynamic structures. IFT is a motile process within flagella in which large protein complexes move from one end of the flagellum to the other. Flagellar length regulation may involve a balance between continuous assembling of tubulin at the tip of the flagellum, mediated by IFT, and continuous disassembling (David et al., 2003). The results of Marshall and Rosembaum (2001) provided support for this idea; these authors found that a partial reduction of the IFT, by either assembly reduction or disassembly increase, leads to a short flagellum (shf) phenotype, and conversely that a long flagellum (lf) mutant shows a decreased rate of microtubule turnover. At this point interesting questions arise: to what extent does the flagellar axoneme turnover? How does such a turnover fit in with mechanisms for flagellar length * Corresponding author. Address: Istituto di Biofisica CNR, Area della Ricerca Pisa, via Alfieri 1, Ghezzano, 56010 Pisa, Italy. Tel.: þ 39-50-3153026; fax: þ 39-50-315-2760. E-mail address: [email protected] (P. Gualtieri). 0968-4328/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2004.01.001
control? To what extent are flagellar length alterations compatible with cell movements? In order to understand how flagellar/ciliary length is regulated, experiments have involved mostly sea urchin embryos (Stephens, 1997) and mutants of unicellular algae such as Chlamydomonas and Dunaliella (Chlorophyta, Chlorophyceae) characterized by flagella of abnormal length. Among these there are the lf mutant, the flagellar assembly defective mutant ( fla), and the shf mutant (Asleson and Lefevbre, 1998; Barsel et al., 1988; Huang et al., 1977; Jarvik et al., 1984; Jarvik et al., 1976; McVittie, 1972). Mutants with short flagella described up to now have been referred to as non-motile cells; this means that short flagella are paralyzed. However, Jarvik (1988) isolated shf mutants of Chlamydomonas that regulate the flagellar length to a mean value of about one-half the length of the wild type. They do not appear defective with respect to cellular or flagellar functions other than length control, since they swim slowly, but with a normal pattern, and display apparently normal photobehavior (Jarvik et al., 1984). In order to explore flagellar size control and its connection with flagellar functionality, we screened Dunaliella salina and Chlamydomonas reihnardtii wild type populations for mutants with a flagellar length less than one-half that of the wild type, and with normal ultrastructural features and normal motility. In this paper we describe a novel, spontaneous mutant of D. salina possessing a pair of stumpy flagella 1 mm long, i.e. much shorter than one-half the length of the wild type, which is 10 – 13 mm. The ultrastructure of
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2. Material and methods
medium. Drops of this suspension were then pipetted onto poly-L -lysine coated coverslips. After dehydration with graded ethanol solutions, the cells were critical point dried, gold coated and observed with a Jeol JSM-5410 scanning electron microscope.
2.1. Cultures
2.6. Immunofluorescence microscopy
Cultures of D. salina wild type and mutant were grown axenically in Johnson medium (1 M NaCl, pH 8.2; Johnson et al., 1968) under constant temperature (24 8C) and continuous illumination (2 £ 102 mmol photons m22 s21).
Four-day old cells were fixed using the cold-methanol method of Deane et al. (2001) with minor modifications. Cells harvested by low speed centrifugation (1000g, 5 min) were fixed in methanol at 2 20 8C for 20 min. Fixed cells were rinsed once in sterile growth medium (pH 8.2), followed by incubation in 5% normal goat serum (NGS) in growth medium for 1 h at 4 8C. Cells were then incubated overnight in a monoclonal anti a-tubulin antibody (clone B5-1-2 Mouse Ascites Fluid, SIGMA) diluted 1:300 in 5% NGS in growth medium. After rinsing, cells were incubated for 2 h at 25 8C in secondary antibody (Molecular Probes Alexa Fluore 350 goat anti-mouse IgG (H þ L) conjugate) diluted 1:300 in 5% NGS in growth medium. Stained cells were examined with a Zeiss Axioplan fluorescence microscope (Zeiss, Germany) equipped with an epifluorescence system, a 100 £ (NA 1.3) planapochromatic objective, and a 100 W mercury lamp. Fluorescence images were acquired with a blue – violet filter set (8-nm band-pass excitation filter, 436 nm; chromatic beam splitter, 460 nm; barrier filter, 470 nm).
these flagella is comparable to that of the wild type, and the mutant shows movement capability and photobehavior.
2.2. Mutant isolation The presence of stumpy mutants in wild type cultures is easily recognizable as a green mat covering the bottom of the flasks. To isolate them, samples taken from these mats were spread on 1.2% (v/v) agar plates and incubated at 24 8C under continuous illumination for 10 days. Individual colonies were then picked from the plates into capillary tubes containing liquid growth medium, and each tube was observed at 40 £ magnification with a dissecting microscope. Colonies without swimming ability were cultured in liquid medium. The mutant phenotype appeared with a frequency of 1026, and reverted to wild type with a frequency of 5 £ 1028. 2.3. Flagellar and cellular length measurements Cells were fixed in methanol at 2 20 8C for 10 min, and rinsed twice in sterile growth medium (pH 8.2). Cell and flagella lengths were measured at 1000 £ using an ocular micrometer and phase contrast optics. Length averages were based on the measurement of 20 replicates for both wild type and mutant cells. 2.4. Transmission electron microscopy Cells from a 4-day old culture were harvested by low speed centrifugation (1000g, 5 min) and fixed at room temperature for 5 min with a mixture of 0.6% glutaraldehyde and 0.5% osmium tetroxide in sterile growth medium (pH 8.2). Fixed cells were washed five times with the same medium (pH 8.2), dehydrated with graded ethanol solutions, and a final step in 100% acetone. Dehydrated cells were incubated overnight in a 1:1 mixture of acetone and EponAraldite resin. The sample was then transferred to 100% resin, and cured in a 60 8C oven for 48 h. Thin sections were obtained with a diamond knife mounted on a LKB ultramicrotome, stained with uranyl acetate and lead citrate and observed in a Jeol 100SX electron microscope. 2.5. Scanning electron microscopy Cells were fixed in 2% osmium solution in sterile growth medium for 5 min and rinsed five times in the same
2.7. Photoaccumulation experiments To verify the presence of photoaccumulation behavior, D. salina wild type and mutant cells were placed in a Petri dish completely covered with black self-sticking paper except for a 2 cm diameter hole on the top of the dish. The two populations were screened for photoaccumulation in liquid medium. A cell concentration of 5 £ 106 cells ml21 was used for both populations. The dishes were illuminated from above with an incandescent lamp (2 £ 102 mmol photons m22 s21). Photographs were taken after 1 h exposure for the wild type and after 15 h for the mutant. The photoaccumulation experiment was performed also on a discontinuous surface consisting of a sintered Pyrexw borosilicate glass filter (40 mm diameter, porosity grade 5, pore index # 10 mm). 2.8. Track reconstruction of Dunaliella mutant movement A Pulnix TM860 (Pulnix, USA) CCD video camera was mounted onto a Zeiss Axioplan microscope (Zeiss, Germany) equipped with 16 £ and 60 £ objectives and 100 W halogen lamp as light source. Cells were placed in a small chamber obtained by fixing a PVC ring onto a microscope slide. The chamber was closed by means of a cover slip so as to avoid sample drying-out. The signal of the camera was the input of a Scion LG-3 Frame Grabber (Scion
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Fig. 1. Schematic drawing of the experimental set-up used for the rotation analysis.
Corporation, USA) plugged into a Pentium III personal computer 750 MHz clock. For the track reconstruction experiment, a sequence of images taken at known intervals of time was acquired, stored, and processed to track the movement of single cells using the automatic procedure of Gualtieri and Coltelli (1991). For the rotation experiment, the light reflected by the cell eyespot was measured. The experimental set-up was the same used for track reconstruction, with the addition of a custom-made slide (Fig. 1). This device allowed the lateral illumination of the cell sample by means of an optical fiber delivering the light coming from a Schott KL1500 fiber optic illuminator (Schott, Germany). 2.9. Photography Photographs were recorded with an Olympus Camedia C-30303 digital camera (Olympus, Japan) mounted on the Zeiss Axioplan microscope (Zeiss, Germany).
3. Results D. salina is a unicellular green alga, ovoid in shape, 8 –9 mm long and 6 –7 mm wide, with an acute apex, tapering anterior, rounded posterior, and two anteriorly inserted equal flagella 10 – 13 mm long (Fig. 2a). The stumpy mutant has the same ovoid shape of the wild type, with slightly different dimension (9 – 10 mm long and 6 –7 mm wide), and the same acute apex where two very short flagella, barely extending outwards but normal in shape, are inserted (Fig. 2b). The flagella of the mutant are about 1 ^ 0.1 mm long from the insertion point on the cell surface to the tip (Fig. 3a). According to the classification by Jarvik and Chojnacki (1985) of stumpy-flagella mutants of Chlamydomonas reinhardtii, our mutant can be considered as belonging to Class 2, i.e. cells with flagella short but otherwise normal. This ‘normality’ was confirmed by comparison of electron microscopy images of thin sections of the mutant (Fig. 3a and b) with corresponding images of the wild type (Fig. 3c and d). In the mutant, axonemal microtubules extend from
Fig. 2. Scanning electron micrograph of the wild type (a) and the stumpy mutant (b). Scale bar: 3 mm.
the basal body (Fig. 3a, BB) to the tip of the stump and are not aberrantly organized. The central doublet (Fig. 3b, CD) is present and extends from the transition zone (Fig. 3a, TZ) to the tip. The axoneme emerges directly from the basal bodies that are interconnected by the striated fibers (also termed distal connecting fibers) (Fig. 3b, SF) and connected to the cytoskeleton by the rootlets (Fig. 3a and b, R). All these structures are organized in a very regular framework and do not differ from those of the wild type (Fig. 3c and d). Another ‘normal’ component of this mutant is the nonaxonemic basal bodies (Leonardi and Ca`ceres, 1994), which invariably appeared in all observed cells (Fig. 3b, NABB). When viewed in cross-section (Fig. 4a and b), the stumpy mutant flagellum shows the typical ‘9 þ 2’ array of microtubules of the wild type (Fig. 4c and d). The central pair of microtubules, the outer and inner dynein arms (Fig. 4a, OD, ID) and the radial spoke (Fig. 4b, RS) are
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Fig. 3. Transmission electron micrographs of longitudinal sections of the apical portion of (a and b) the mutant, (c and d) the wild type. Refer to the text for details. CD, central doublet; NABB, non-axonemic basal body; BB, basal body; R, rootlet; SF, striated fiber; TZ, transition zone. Scale bar: 1 mm.
Fig. 4. Transmission electron micrographs of transverse sections of (a and b) the mutant flagellum; (c and d) the wild type flagellum. Refer to the text for details. ID, inner dynein arms; OD, outer dynein arms; RS, radial spokes. Scale bar: 100 nm.
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easily recognizable. No electron-opaque material and/or amorphous material surround the microtubules. In both the mutant and the wild type, all around the plasmalemma, especially in the flagellar zone, an extensive dense coat, sometimes fuzzy, is visible, confirming the presence of a glycocalyx-type cell envelope (Oliviera et al., 1980; Figs. 3 and 4). The normality of the mutant is confirmed also by its growth curve, which can be considered superimposable to that of the wild type for all the phases (data not shown). To verify that the mutation did not damage the microtubular system of the flagella and that underlying the plasmalemma, immunofluorescence analysis was performed on both the mutant (Fig. 5a) and the wild type (Fig. 5b). In
Fig. 5. Immunofluorescence localization of tubulin in the mutant (a) and the wild type (b). Arrowheads point to the axoneme and cytoskeletal microtubules.
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the mutant stained with the monoclonal antibody against atubulin, the cytoplasmic microtubules and those of the axonemes are clearly visible (arrowheads, Fig. 5a and b). Under optical microscopy, mutant cells showed small, irregular oscillations on the plane of the slide, similar to the movements of a swinging lever hinged at one side; no flagellar movements could be appreciated due to the short length of the stumpy flagella. To analyze the movements of the mutant, we set-up a digital procedure that stores the elapsed frames of microscope fields acquired in transmitted light into the computer memory. Frames were acquired every 200 ms. The results are reported in Fig. 6. Six selected images are shown. The total elapsed time is 37 min. Each cell is numbered and can be followed in the successive frame where its trajectory is represented with a black line starting from the previous cell barycentre. Since cell trajectories are randomly directed, we can say that the cells move and are not transported by a flow in the medium. The calculated average velocity of the mutant is 0.2 mm s21. Tracking experiment was performed also on wild type cells (Fig. 7). In this case frames were acquired every 40 ms, for a total elapsed time of 400 ms. Six selected images are shown. Cell trajectories are not oriented, and indicate an active, straight swimming of the cells. The calculated average velocity of the wild type is 95 mm s21. To further investigate which kind of movement the mutant performs, another digital procedure was used, which can store in the computer memory the frames acquired under lateral illumination (Fig. 1). The eyespot of Chlorophyta such as Chlamydomonas and Dunaliella is a quarter-wavelength multi-layered organization of osmophilic granules, which reflects very efficiently the light that strikes upon it. As the cell moves, we can detect this brilliant spot and verify if the cell rotates or not. For the mutant frames were acquired every 200 ms, for a total of 2000 frames. The 40 frames shown in Fig. 8 represent every 50th frame. The total elapsed time between the first and the last frame is 400 s. The result of the recording shows that mutant cells do not rotate around their longitudinal axis but seem to oscillate irregularly around it (Fig. 8). The mutant is capable of sensing the light and responding somehow to its direction. For the wild type frames were acquired every 40 ms. The image resulting from a 500 ms recording show that these cells rotate twice a second (Fig. 9). In the photoaccumulation experiment both the mutant and the wild type show a similar behavior, the main difference being the time necessary to accumulate. Fig. 10 shows the result of the photoaccumulation of the wild type after 1 h (Fig. 10a), and of the mutant after 1 h (Fig. 10b), and after 15 h (Fig. 10c); the density profile of the cell population is presented for each Petri dish. In the case of the wild type, the density profile after 1 h (Fig. 10a) shows a sharp peak corresponding to the accumulated cells in the illuminated spot of the dish; the flatness of the peak indicates the homogeneity of the phenomenon. As to the mutant, despite its extremely low
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Fig. 6. Result of the track reconstruction experiment for the mutant. The black lines represent the cell trajectories. The total elapsed time between the first and the last image is 37 min.
velocity, after 1 h (Fig. 10b) the density profile shows a peak, though lower than that of the wild type, in correspondence to the illuminated spot. Fifteen hours are necessary for the mutant to achieve an accumulation peak comparable to that of the wild type (Fig. 10c); the corresponding profile indicates that the population is moving from the periphery of the dish towards the accumulation spot. When the photoaccumulation experiment was performed on a discontinuous surface, the mutant showed no accumulation, whereas the wild type showed a normal accumulation pattern (data not shown).
4. Discussion Our results suggest that in Dunaliella the length of a normal flagellum can be regulated at any level, ‘from toe to
tip’, above the transition zone. Assuming that the IFT is the process that controls the flagellar length, this length is regulated by the balance between a length-dependent assembly rate, with a transport of flagellar component as limiting step, and a length-independent disassembly rate. As reported by Jarvik et al. (1984), a mutant of C. reinhardtii with flagellar length of a mean value of about one-half the length of the wild type, and with no defects of the cellular or flagellar functions other than length control, can swim slowly but with a normal pattern. Our mutant of Dunaliella cannot swim, since its flagellum, though not structurally defective, is not capable of producing and propagating a bending wave along the flagellum due to its extremely reduced length. Still, the mutant can move as shown by the trajectory reconstruction experiment (Fig. 6), and can follow light stimuli as shown by the photoaccumulation experiments (Fig. 10). A possible
Fig. 7. Result of the track reconstruction experiment for the wild type. The black lines represent the cell trajectories. The total elapsed time between the first and the last image is 400 ms.
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Fig. 8. Result of the rotation experiment for the mutant. Each image represents the sampling of one every 50th frame, for a total elapsed time of 400 s.
explanation for this motion, is that the mutant performs gliding, whereby the contact with a solid substrate (e.g. the glass of a microscope slide) draws the cell along the surface. In fact, when the experiment was performed by placing the cells on the discontinuous surface of a porous glass filter, no accumulation was detected, since the cells lacked the substrate necessary for the gliding motion. By means of the trajectories reconstruction, we can calculate the velocity of the mutant. The figure is very low (0.2 mm s21) and for this reason it is impossible to detect any displacement under the microscope. It is well known that Chlamydomonas possesses
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two separate mechanisms for cell motility, both involving the flagella. Cell may either swim or glide by means of flagellar surface – substrate interactions (Bloodgood, 1988; Lewin, 1982). The motors for swimming motility and gliding motility are different. In gliding motility the proteins of the flagellar membrane mechanically interact with the substrate; force applied to these attached proteins results in their lateral movement in the plane of the flagellar membrane and the subsequent movement of the cell relative to the substrate. This requires the mechanical coupling of the axoneme outer doublet microtubules to the flagellar membrane proteins, and the presence of the microtubule sliding mechanism (Brokaw, 1989; Brokaw and Johnson, 1989). On the basis of both the immunofluorescence and electron microscopy images, we can say that the flagellum of our mutant possesses all the machinery required to generate its own movement, though it is too short to generate a bend. Hence, it should retain also the microtubule sliding mechanism necessary for the gliding. The difference between gliding and swimming movements is confirmed by the result of the reflection experiments (Figs. 8 and 9). Wild type cells of Dunaliella rotate during their motion with a frequency of twice a second around their longitudinal axis and this rotation can be easily tracked by following the bright spot of the eyespot on either side of the cell. In the case of the mutant, the eyespot is visible always on the same side of the cell indicating that the cell does not rotate. It is more difficult to explain how the mutant can orient in a luminous field. The motion pattern of the wild type allows the sequential sampling of the environment to determine the orientation of the light field: the cells measure directly a temporal gradient, infer a spatial gradient from the information on the movement of the receptor and orient accordingly. The motion of the mutant lacks the coherent pattern of movement along a sinusoidal path of the wild type (Fig. 9). It moves with a biased random walk (Fig. 6), hence its nearing the stimulus direction is indirect, and by chance. According to Schoevaert et al. (1988) the two Dunaliella flagella, though possessing the same structure, and being symmetrical in relation to the antero-posterior plane of the cell, do not have the same behavior. Unlike the flagella of Chlamydomonas, whose different intrinsic beat frequencies are not expressed in free-swimming cells (Takada and Kamiya, 1997), the two flagella of Dunaliella do not have the same frequency during forward motion, and when the cell changes direction, one flagellum continues to beat,
Fig. 9. Result of the rotation experiment for the wild type. The frames were acquired every 40 ms.
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Fig. 10. Result of the photoaccumulation experiment: the three Petri dishes show the accumulation of the wild type (a) and the mutant (b) after 1 h, and the mutant after 15 h (c). Above each dish, the corresponding density profile is presented.
while the other one is inactivated. Therefore, Dunaliella can control the direction of its path quite efficiently, and this ability could be particularly useful for the gliding movement of the mutant. A possible explanation of the motion of these cells is that they scramble over the surface by alternately oscillating the body to the left and to the right as the clapper of a bell, and pivoting up and down along a small arch, hinging upon the left or the right stump.
References Asleson, C.M., Lefevbre, P.A., 1998. Genetic analysis of flagellar length control in Chlamydomonas reinhardtii: a new long-flagella locus and extragenic suppressor mutations. Genetics 148, 693–702. Barsel, S.E., Wexler, D.E., Lefevbre, P.A., 1988. Genetic analysis of longflagella mutants of Chlamydomonas reinhardtii. Genetics 118, 637– 648. Bloodgood, R.A., 1988. Gliding motility and the dynamics of flagellar membrane glycoproteins in Chlamydomonas reinhardtii. J. Protozool. 35 (4), 552 –558. Brokaw, C.J., 1989. Direct measurements of sliding between outer doublet microtubules in swimming sperm flagella. Science 243, 1593–1596. Brokaw, C.J., Johnson, K.A., 1989. Dynein-induced microtubule sliding and force generation. In: Warner, F., Satir, P., Gibbons, R. (Eds.), Cell Movement, The Dynein ATPases, vol. 1. A.R. Liss, New York, pp. 191– 198.
David, J., Parker, K., Quarmby, L.M., 2003. Chlamydomonas fla mutants reveal a link between deflagellation and intraflagellar transport. BMC Cell Biol. 4, 11–22. Deane, J.A., Cole, D.G., Seeley, E.S., Diener, D.R., Rosembaum, J.L., 2001. Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibres as the docking site for IFT particles. Curr. Biol. 11, 1586–1590. Gualtieri, P., Coltelli, P., 1991. A real-time automated system for the analysis of moving images. J. Compute-Assist. Microsc. 3 (1), 15 –21. Huang, B., Rifkin, M.R., Luck, D.J., 1977. Temperature-sensitive mutations affecting flagellar assembly and function in Chlamydomonas reinhardtii. J. Cell Biol. 72, 67–85. Jarvik, W.J., 1988. Size-control in the Chlamydomonas reinhardtii flagellum. J. Protozool. 35 (4), 570–573. Jarvik, W.J., Chojnacki, B., 1985. Flagellar morphology in stumpy-flagella mutants of Chlamydomonas reinhardtii. J. Protozool. 32 (4), 649 –656. Jarvik, W.J., Lefebvre, P.W., Rosembaum, J.L., 1976. A cold-sensitive mutant of Chlamydomsonas reinhardtii with aberrant control of flagellar length. J. Cell Biol. 70, 149a. Jarvik, W.J., Reinhart, F.D., Kuchka, M.R., Adler, S.A., 1984. Altered flagellar size-control in shf-1 short-flagella mutants of Chlamydomonas reinhardtii. J. Protozool. 31 (2), 199 –204. Johnson, M.K., Johnson, E.J., MacElroy, R.D., Speer, H.L., Bruff, B.S., 1968. Effects of salts on the halophilic alga Dunaliella viridis. Am. Soc. Microbiol. 95 (4), 1461–1468. Leonardi, P.I., Ca`ceres, E.J., 1994. Comparative analysis of the fine structure of young and adult individuals of Dunaliella salina (Polyblepharidaceae, Chlorophyceae) with emphasis on the flagellar apparatus. J. Phycol. 30, 642– 653. Lewin, R.A., 1982. A new kind of motility mutant (non-gliding) in Chlamydomonas. Experientia 38, 348–349. Marshall, W.F., Rosembaum, J.L., 2001. Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control. J. Cell Biol. 155 (3), 405 –414. McVittie, A.C., 1972. Flagellum mutants of Chlamydomonas reinnhardtii. J. Gen. Microbiol. 71, 525 –540. Oliviera, L., Bisalputra, T., Antia, N.J., 1980. Ultrastructural observation of the surface coat of Dunaliella tertiolecta from staining with cationic dyes and enzyme treatments. New Phytol. 85, 385–392. Schoevaert, D., Krishnaswamy, S., Couturier, M., Marano, F., 1988. Ciliary beat and cell motility of Dunaliella: computer analysis of high speed microcinematography. Biol. Cell 62, 229 –240. Stephens, R.E., 1997. Synthesis and turnover of embryonic sea urchin ciliary proteins during selective inhibition of tubulin synthesis and assembly. Mol. Biol. Cell. 8 (11), 2187–2198. Takada, S., Kamiya, R., 1997. Beat frequency difference between the two flagella of Chlamydomonas depends on the attachment site of outer dynein arms on the outer-doublet microtubules. Cell Motil. Cytoskeleton 36, 68–75.
A short flagella mutant of Dunaliella salina (Chlorophyta, Chlorophyceae) Rosa Vismaraa, Franco Vernib, Laura Barsantia, Valtere Evangelistaa, Paolo Gualtieria,* a Istituto di Biofisica CNR, Area della Ricerca Pisa, Via Moruzzi 1, 56124 Pisa, Italy Dipartimento di Etologia, Ecologia, Evoluzione, Universita` degli Studi di Pisa, Via A. Volta 6, 56127 Pisa, Italy
b
Abstract Dunaliella salina (Chlorophyta, Chlorophyceae) is a unicellular wall-less biflagellate alga. In this paper we describe a spontaneous mutant of D. salina, isolated from wild type cultures, which is characterized by very short flagella. The ultrastructure showed the basic 9 þ 2 organization of wild-type flagella. Immunofluorescence localization of tubulin in this mutant confirmed the normal construction of the axoneme. Although, the mutant does not swim, still it is able to move and perform photobehavior. As shown by track reconstruction, and rotation movements, observed by means of reflection microscopy, this mutant can move, probably gliding by means of its stumpy flagella. A possible model to explain the mutant motion pattern is discussed. q 2004 Elsevier Ltd. All rights reserved. Keywords: Mutant; Photobehaviour; Track reconstruction; Flagella; Gliding
1. Introduction The mechanisms that determine and preserve the size and function of cellular organelles represent a fundamental question in cell biology, which up to now has been only partially understood. Cilia and flagella, in metazoan as well as in unicellular organisms, provide a handy model system to investigate size-control analysis of organelles. The phenomenon of intraflagellar transport (IFT) provides evidence that flagella are dynamic structures. IFT is a motile process within flagella in which large protein complexes move from one end of the flagellum to the other. Flagellar length regulation may involve a balance between continuous assembling of tubulin at the tip of the flagellum, mediated by IFT, and continuous disassembling (David et al., 2003). The results of Marshall and Rosembaum (2001) provided support for this idea; these authors found that a partial reduction of the IFT, by either assembly reduction or disassembly increase, leads to a short flagellum (shf) phenotype, and conversely that a long flagellum (lf) mutant shows a decreased rate of microtubule turnover. At this point interesting questions arise: to what extent does the flagellar axoneme turnover? How does such a turnover fit in with mechanisms for flagellar length * Corresponding author. Address: Istituto di Biofisica CNR, Area della Ricerca Pisa, via Alfieri 1, Ghezzano, 56010 Pisa, Italy. Tel.: þ 39-50-3153026; fax: þ 39-50-315-2760. E-mail address: [email protected] (P. Gualtieri). 0968-4328/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2004.01.001
control? To what extent are flagellar length alterations compatible with cell movements? In order to understand how flagellar/ciliary length is regulated, experiments have involved mostly sea urchin embryos (Stephens, 1997) and mutants of unicellular algae such as Chlamydomonas and Dunaliella (Chlorophyta, Chlorophyceae) characterized by flagella of abnormal length. Among these there are the lf mutant, the flagellar assembly defective mutant ( fla), and the shf mutant (Asleson and Lefevbre, 1998; Barsel et al., 1988; Huang et al., 1977; Jarvik et al., 1984; Jarvik et al., 1976; McVittie, 1972). Mutants with short flagella described up to now have been referred to as non-motile cells; this means that short flagella are paralyzed. However, Jarvik (1988) isolated shf mutants of Chlamydomonas that regulate the flagellar length to a mean value of about one-half the length of the wild type. They do not appear defective with respect to cellular or flagellar functions other than length control, since they swim slowly, but with a normal pattern, and display apparently normal photobehavior (Jarvik et al., 1984). In order to explore flagellar size control and its connection with flagellar functionality, we screened Dunaliella salina and Chlamydomonas reihnardtii wild type populations for mutants with a flagellar length less than one-half that of the wild type, and with normal ultrastructural features and normal motility. In this paper we describe a novel, spontaneous mutant of D. salina possessing a pair of stumpy flagella 1 mm long, i.e. much shorter than one-half the length of the wild type, which is 10 – 13 mm. The ultrastructure of
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2. Material and methods
medium. Drops of this suspension were then pipetted onto poly-L -lysine coated coverslips. After dehydration with graded ethanol solutions, the cells were critical point dried, gold coated and observed with a Jeol JSM-5410 scanning electron microscope.
2.1. Cultures
2.6. Immunofluorescence microscopy
Cultures of D. salina wild type and mutant were grown axenically in Johnson medium (1 M NaCl, pH 8.2; Johnson et al., 1968) under constant temperature (24 8C) and continuous illumination (2 £ 102 mmol photons m22 s21).
Four-day old cells were fixed using the cold-methanol method of Deane et al. (2001) with minor modifications. Cells harvested by low speed centrifugation (1000g, 5 min) were fixed in methanol at 2 20 8C for 20 min. Fixed cells were rinsed once in sterile growth medium (pH 8.2), followed by incubation in 5% normal goat serum (NGS) in growth medium for 1 h at 4 8C. Cells were then incubated overnight in a monoclonal anti a-tubulin antibody (clone B5-1-2 Mouse Ascites Fluid, SIGMA) diluted 1:300 in 5% NGS in growth medium. After rinsing, cells were incubated for 2 h at 25 8C in secondary antibody (Molecular Probes Alexa Fluore 350 goat anti-mouse IgG (H þ L) conjugate) diluted 1:300 in 5% NGS in growth medium. Stained cells were examined with a Zeiss Axioplan fluorescence microscope (Zeiss, Germany) equipped with an epifluorescence system, a 100 £ (NA 1.3) planapochromatic objective, and a 100 W mercury lamp. Fluorescence images were acquired with a blue – violet filter set (8-nm band-pass excitation filter, 436 nm; chromatic beam splitter, 460 nm; barrier filter, 470 nm).
these flagella is comparable to that of the wild type, and the mutant shows movement capability and photobehavior.
2.2. Mutant isolation The presence of stumpy mutants in wild type cultures is easily recognizable as a green mat covering the bottom of the flasks. To isolate them, samples taken from these mats were spread on 1.2% (v/v) agar plates and incubated at 24 8C under continuous illumination for 10 days. Individual colonies were then picked from the plates into capillary tubes containing liquid growth medium, and each tube was observed at 40 £ magnification with a dissecting microscope. Colonies without swimming ability were cultured in liquid medium. The mutant phenotype appeared with a frequency of 1026, and reverted to wild type with a frequency of 5 £ 1028. 2.3. Flagellar and cellular length measurements Cells were fixed in methanol at 2 20 8C for 10 min, and rinsed twice in sterile growth medium (pH 8.2). Cell and flagella lengths were measured at 1000 £ using an ocular micrometer and phase contrast optics. Length averages were based on the measurement of 20 replicates for both wild type and mutant cells. 2.4. Transmission electron microscopy Cells from a 4-day old culture were harvested by low speed centrifugation (1000g, 5 min) and fixed at room temperature for 5 min with a mixture of 0.6% glutaraldehyde and 0.5% osmium tetroxide in sterile growth medium (pH 8.2). Fixed cells were washed five times with the same medium (pH 8.2), dehydrated with graded ethanol solutions, and a final step in 100% acetone. Dehydrated cells were incubated overnight in a 1:1 mixture of acetone and EponAraldite resin. The sample was then transferred to 100% resin, and cured in a 60 8C oven for 48 h. Thin sections were obtained with a diamond knife mounted on a LKB ultramicrotome, stained with uranyl acetate and lead citrate and observed in a Jeol 100SX electron microscope. 2.5. Scanning electron microscopy Cells were fixed in 2% osmium solution in sterile growth medium for 5 min and rinsed five times in the same
2.7. Photoaccumulation experiments To verify the presence of photoaccumulation behavior, D. salina wild type and mutant cells were placed in a Petri dish completely covered with black self-sticking paper except for a 2 cm diameter hole on the top of the dish. The two populations were screened for photoaccumulation in liquid medium. A cell concentration of 5 £ 106 cells ml21 was used for both populations. The dishes were illuminated from above with an incandescent lamp (2 £ 102 mmol photons m22 s21). Photographs were taken after 1 h exposure for the wild type and after 15 h for the mutant. The photoaccumulation experiment was performed also on a discontinuous surface consisting of a sintered Pyrexw borosilicate glass filter (40 mm diameter, porosity grade 5, pore index # 10 mm). 2.8. Track reconstruction of Dunaliella mutant movement A Pulnix TM860 (Pulnix, USA) CCD video camera was mounted onto a Zeiss Axioplan microscope (Zeiss, Germany) equipped with 16 £ and 60 £ objectives and 100 W halogen lamp as light source. Cells were placed in a small chamber obtained by fixing a PVC ring onto a microscope slide. The chamber was closed by means of a cover slip so as to avoid sample drying-out. The signal of the camera was the input of a Scion LG-3 Frame Grabber (Scion
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Fig. 1. Schematic drawing of the experimental set-up used for the rotation analysis.
Corporation, USA) plugged into a Pentium III personal computer 750 MHz clock. For the track reconstruction experiment, a sequence of images taken at known intervals of time was acquired, stored, and processed to track the movement of single cells using the automatic procedure of Gualtieri and Coltelli (1991). For the rotation experiment, the light reflected by the cell eyespot was measured. The experimental set-up was the same used for track reconstruction, with the addition of a custom-made slide (Fig. 1). This device allowed the lateral illumination of the cell sample by means of an optical fiber delivering the light coming from a Schott KL1500 fiber optic illuminator (Schott, Germany). 2.9. Photography Photographs were recorded with an Olympus Camedia C-30303 digital camera (Olympus, Japan) mounted on the Zeiss Axioplan microscope (Zeiss, Germany).
3. Results D. salina is a unicellular green alga, ovoid in shape, 8 –9 mm long and 6 –7 mm wide, with an acute apex, tapering anterior, rounded posterior, and two anteriorly inserted equal flagella 10 – 13 mm long (Fig. 2a). The stumpy mutant has the same ovoid shape of the wild type, with slightly different dimension (9 – 10 mm long and 6 –7 mm wide), and the same acute apex where two very short flagella, barely extending outwards but normal in shape, are inserted (Fig. 2b). The flagella of the mutant are about 1 ^ 0.1 mm long from the insertion point on the cell surface to the tip (Fig. 3a). According to the classification by Jarvik and Chojnacki (1985) of stumpy-flagella mutants of Chlamydomonas reinhardtii, our mutant can be considered as belonging to Class 2, i.e. cells with flagella short but otherwise normal. This ‘normality’ was confirmed by comparison of electron microscopy images of thin sections of the mutant (Fig. 3a and b) with corresponding images of the wild type (Fig. 3c and d). In the mutant, axonemal microtubules extend from
Fig. 2. Scanning electron micrograph of the wild type (a) and the stumpy mutant (b). Scale bar: 3 mm.
the basal body (Fig. 3a, BB) to the tip of the stump and are not aberrantly organized. The central doublet (Fig. 3b, CD) is present and extends from the transition zone (Fig. 3a, TZ) to the tip. The axoneme emerges directly from the basal bodies that are interconnected by the striated fibers (also termed distal connecting fibers) (Fig. 3b, SF) and connected to the cytoskeleton by the rootlets (Fig. 3a and b, R). All these structures are organized in a very regular framework and do not differ from those of the wild type (Fig. 3c and d). Another ‘normal’ component of this mutant is the nonaxonemic basal bodies (Leonardi and Ca`ceres, 1994), which invariably appeared in all observed cells (Fig. 3b, NABB). When viewed in cross-section (Fig. 4a and b), the stumpy mutant flagellum shows the typical ‘9 þ 2’ array of microtubules of the wild type (Fig. 4c and d). The central pair of microtubules, the outer and inner dynein arms (Fig. 4a, OD, ID) and the radial spoke (Fig. 4b, RS) are
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Fig. 3. Transmission electron micrographs of longitudinal sections of the apical portion of (a and b) the mutant, (c and d) the wild type. Refer to the text for details. CD, central doublet; NABB, non-axonemic basal body; BB, basal body; R, rootlet; SF, striated fiber; TZ, transition zone. Scale bar: 1 mm.
Fig. 4. Transmission electron micrographs of transverse sections of (a and b) the mutant flagellum; (c and d) the wild type flagellum. Refer to the text for details. ID, inner dynein arms; OD, outer dynein arms; RS, radial spokes. Scale bar: 100 nm.
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easily recognizable. No electron-opaque material and/or amorphous material surround the microtubules. In both the mutant and the wild type, all around the plasmalemma, especially in the flagellar zone, an extensive dense coat, sometimes fuzzy, is visible, confirming the presence of a glycocalyx-type cell envelope (Oliviera et al., 1980; Figs. 3 and 4). The normality of the mutant is confirmed also by its growth curve, which can be considered superimposable to that of the wild type for all the phases (data not shown). To verify that the mutation did not damage the microtubular system of the flagella and that underlying the plasmalemma, immunofluorescence analysis was performed on both the mutant (Fig. 5a) and the wild type (Fig. 5b). In
Fig. 5. Immunofluorescence localization of tubulin in the mutant (a) and the wild type (b). Arrowheads point to the axoneme and cytoskeletal microtubules.
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the mutant stained with the monoclonal antibody against atubulin, the cytoplasmic microtubules and those of the axonemes are clearly visible (arrowheads, Fig. 5a and b). Under optical microscopy, mutant cells showed small, irregular oscillations on the plane of the slide, similar to the movements of a swinging lever hinged at one side; no flagellar movements could be appreciated due to the short length of the stumpy flagella. To analyze the movements of the mutant, we set-up a digital procedure that stores the elapsed frames of microscope fields acquired in transmitted light into the computer memory. Frames were acquired every 200 ms. The results are reported in Fig. 6. Six selected images are shown. The total elapsed time is 37 min. Each cell is numbered and can be followed in the successive frame where its trajectory is represented with a black line starting from the previous cell barycentre. Since cell trajectories are randomly directed, we can say that the cells move and are not transported by a flow in the medium. The calculated average velocity of the mutant is 0.2 mm s21. Tracking experiment was performed also on wild type cells (Fig. 7). In this case frames were acquired every 40 ms, for a total elapsed time of 400 ms. Six selected images are shown. Cell trajectories are not oriented, and indicate an active, straight swimming of the cells. The calculated average velocity of the wild type is 95 mm s21. To further investigate which kind of movement the mutant performs, another digital procedure was used, which can store in the computer memory the frames acquired under lateral illumination (Fig. 1). The eyespot of Chlorophyta such as Chlamydomonas and Dunaliella is a quarter-wavelength multi-layered organization of osmophilic granules, which reflects very efficiently the light that strikes upon it. As the cell moves, we can detect this brilliant spot and verify if the cell rotates or not. For the mutant frames were acquired every 200 ms, for a total of 2000 frames. The 40 frames shown in Fig. 8 represent every 50th frame. The total elapsed time between the first and the last frame is 400 s. The result of the recording shows that mutant cells do not rotate around their longitudinal axis but seem to oscillate irregularly around it (Fig. 8). The mutant is capable of sensing the light and responding somehow to its direction. For the wild type frames were acquired every 40 ms. The image resulting from a 500 ms recording show that these cells rotate twice a second (Fig. 9). In the photoaccumulation experiment both the mutant and the wild type show a similar behavior, the main difference being the time necessary to accumulate. Fig. 10 shows the result of the photoaccumulation of the wild type after 1 h (Fig. 10a), and of the mutant after 1 h (Fig. 10b), and after 15 h (Fig. 10c); the density profile of the cell population is presented for each Petri dish. In the case of the wild type, the density profile after 1 h (Fig. 10a) shows a sharp peak corresponding to the accumulated cells in the illuminated spot of the dish; the flatness of the peak indicates the homogeneity of the phenomenon. As to the mutant, despite its extremely low
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Fig. 6. Result of the track reconstruction experiment for the mutant. The black lines represent the cell trajectories. The total elapsed time between the first and the last image is 37 min.
velocity, after 1 h (Fig. 10b) the density profile shows a peak, though lower than that of the wild type, in correspondence to the illuminated spot. Fifteen hours are necessary for the mutant to achieve an accumulation peak comparable to that of the wild type (Fig. 10c); the corresponding profile indicates that the population is moving from the periphery of the dish towards the accumulation spot. When the photoaccumulation experiment was performed on a discontinuous surface, the mutant showed no accumulation, whereas the wild type showed a normal accumulation pattern (data not shown).
4. Discussion Our results suggest that in Dunaliella the length of a normal flagellum can be regulated at any level, ‘from toe to
tip’, above the transition zone. Assuming that the IFT is the process that controls the flagellar length, this length is regulated by the balance between a length-dependent assembly rate, with a transport of flagellar component as limiting step, and a length-independent disassembly rate. As reported by Jarvik et al. (1984), a mutant of C. reinhardtii with flagellar length of a mean value of about one-half the length of the wild type, and with no defects of the cellular or flagellar functions other than length control, can swim slowly but with a normal pattern. Our mutant of Dunaliella cannot swim, since its flagellum, though not structurally defective, is not capable of producing and propagating a bending wave along the flagellum due to its extremely reduced length. Still, the mutant can move as shown by the trajectory reconstruction experiment (Fig. 6), and can follow light stimuli as shown by the photoaccumulation experiments (Fig. 10). A possible
Fig. 7. Result of the track reconstruction experiment for the wild type. The black lines represent the cell trajectories. The total elapsed time between the first and the last image is 400 ms.
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Fig. 8. Result of the rotation experiment for the mutant. Each image represents the sampling of one every 50th frame, for a total elapsed time of 400 s.
explanation for this motion, is that the mutant performs gliding, whereby the contact with a solid substrate (e.g. the glass of a microscope slide) draws the cell along the surface. In fact, when the experiment was performed by placing the cells on the discontinuous surface of a porous glass filter, no accumulation was detected, since the cells lacked the substrate necessary for the gliding motion. By means of the trajectories reconstruction, we can calculate the velocity of the mutant. The figure is very low (0.2 mm s21) and for this reason it is impossible to detect any displacement under the microscope. It is well known that Chlamydomonas possesses
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two separate mechanisms for cell motility, both involving the flagella. Cell may either swim or glide by means of flagellar surface – substrate interactions (Bloodgood, 1988; Lewin, 1982). The motors for swimming motility and gliding motility are different. In gliding motility the proteins of the flagellar membrane mechanically interact with the substrate; force applied to these attached proteins results in their lateral movement in the plane of the flagellar membrane and the subsequent movement of the cell relative to the substrate. This requires the mechanical coupling of the axoneme outer doublet microtubules to the flagellar membrane proteins, and the presence of the microtubule sliding mechanism (Brokaw, 1989; Brokaw and Johnson, 1989). On the basis of both the immunofluorescence and electron microscopy images, we can say that the flagellum of our mutant possesses all the machinery required to generate its own movement, though it is too short to generate a bend. Hence, it should retain also the microtubule sliding mechanism necessary for the gliding. The difference between gliding and swimming movements is confirmed by the result of the reflection experiments (Figs. 8 and 9). Wild type cells of Dunaliella rotate during their motion with a frequency of twice a second around their longitudinal axis and this rotation can be easily tracked by following the bright spot of the eyespot on either side of the cell. In the case of the mutant, the eyespot is visible always on the same side of the cell indicating that the cell does not rotate. It is more difficult to explain how the mutant can orient in a luminous field. The motion pattern of the wild type allows the sequential sampling of the environment to determine the orientation of the light field: the cells measure directly a temporal gradient, infer a spatial gradient from the information on the movement of the receptor and orient accordingly. The motion of the mutant lacks the coherent pattern of movement along a sinusoidal path of the wild type (Fig. 9). It moves with a biased random walk (Fig. 6), hence its nearing the stimulus direction is indirect, and by chance. According to Schoevaert et al. (1988) the two Dunaliella flagella, though possessing the same structure, and being symmetrical in relation to the antero-posterior plane of the cell, do not have the same behavior. Unlike the flagella of Chlamydomonas, whose different intrinsic beat frequencies are not expressed in free-swimming cells (Takada and Kamiya, 1997), the two flagella of Dunaliella do not have the same frequency during forward motion, and when the cell changes direction, one flagellum continues to beat,
Fig. 9. Result of the rotation experiment for the wild type. The frames were acquired every 40 ms.
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Fig. 10. Result of the photoaccumulation experiment: the three Petri dishes show the accumulation of the wild type (a) and the mutant (b) after 1 h, and the mutant after 15 h (c). Above each dish, the corresponding density profile is presented.
while the other one is inactivated. Therefore, Dunaliella can control the direction of its path quite efficiently, and this ability could be particularly useful for the gliding movement of the mutant. A possible explanation of the motion of these cells is that they scramble over the surface by alternately oscillating the body to the left and to the right as the clapper of a bell, and pivoting up and down along a small arch, hinging upon the left or the right stump.
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