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Address correspondence to Ke Hu, University of Pennsylvania, Dept. of Biology, 305 Goddard Laboratories, Philadelphia, PA 19104-6018. Tel.: (215) 898-2120. Fax: (215) 746-6697. E-mail: huke{at}mail.sas.upenn.edu
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Abstract |
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Key Words: Apicomplexan parasites; microtubules; motility; cytoskeleton; cryoelectron microscopy
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Introduction |
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Many different polymeric arrangements of tubulin occur in vivo, and an even larger number can be induced to form in vitro (Dustin, 1984; Murray, 1991). All the various arrangements presumably share a relatively small set of bonding patterns between subunits, thus accounting for the shared symmetry elements apparent in their architecture. These bonding preferences lead to straight chains of tubulin dimers associated end to end (protofilaments), which in turn are associated side by side into curved sheets of parallel columns. In cross-section, the profile of the various structures built from these curved sheets is a circle or an arc of a circle, as expected if all the protofilamentprotofilament interactions are identical. Profiles lacking circular symmetry have not been described.
Here we report that the conoid fibers of T. gondii are assembled by a rapid incorporation of tubulin subunits during early but not late stages of cell division, and that in the mature conoid, tubulin is arranged into a novel polymer form that is quite different from typical microtubules.
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Results |
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The presence of tubulin in the apical complex was confirmed by indirect immunofluorescence with monoclonal antibodies against - and ß-tubulin, as shown in Fig. 2. Anti-tubulin antibodies label only the subpellicular microtubules in cells treated as for normal immunocytochemical labeling, as previously reported (Schwartzman et al., 1985). However, more vigorous detergent treatment, or treatment with detergent plus high salt extraction, permitted labeling of the conoid. Anti-tubulin strongly labels both the 22 subpellicular microtubules and the apical end of the parasites, colocalizing with the structures containing YFP (Fig. 2, AC). Western blot analysis of whole parasite extracts demonstrates YFP antibody reactivity with full-length YFP-
-tubulin only. No other form of YFP is present in detectable amount (Fig. 2 D).
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The conoid fibers are composed of tubulin
The apical elements under study are 0.3 µm long by 0.4 µm wide, too small to be well resolved by light microscopy. Electron microscopic images of thin sections of the apical region (Fig. 4) reveal several distinctive organelles, including the conoid, polar rings, rhoptries, and intraconoid microtubules (Scholtyseck et al., 1970; de Souza, 1974; Russell and Burns, 1984; Nichols and Chiappino, 1987; Dubremetz et al., 1989; Carruthers and Sibley, 1997). The conoid is a truncated cone
280 nm long (Fig. 4 A). Transverse thin sections through the apical end of parasites treated with tannic acid show the conoid as a 380-nm diameter ring of electron dense material (Fig. 4 C). The conoid fibers give rise to a set of spiral markings modulating the density of this ring. Outside the conoid, subpellicular microtubules emanate from the lower polar ring (postulated to be a microtubule-organizing center; Russell and Burns, 1984) spiraling outward and towards the posterior end of the parasite (Fig. 4 C, arrows). Inside the conoid, the two intraconoid microtubules are clearly visible, exhibiting canonical microtubule structure (a circle of 13 protofilaments). In the image shown, these two microtubules are sandwiched between two rhoptries, membrane-bounded secretory organelles believed to be involved in host-cell invasion. Glancing sections sometimes include a single wall of the conoid, in which the highly curved conoid fibers are visible over a short distance (Fig. 4 B). Faint longitudinal striations can be seen running along the fibers.
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Tubulin in the conoid fibers is assembled into protofilaments
To further characterize these tubulin containing fibers, Toxoplasma was examined by electron microscopy using specimens prepared in several different ways, including negative staining of detergent extracted cytoskeletons with phosphotungstic acid, cryoelectron microscopy of unfixed, unstained, frozen-hydrated cytoskeletons, and longitudinal and cross-sections of fixed, embedded parasites (both intact and detergent extracted).
In thin sagittal sections, the walls of the conoid appear as two vertical bands of electron-dense material 280 nm long and 35 nm wide (Figs. 4 A and 6, A and B). The conoid fibers are embedded in this dense matrix, and in appropriately oriented regions they can be seen in cross section. If the plane of section is parallel to the axis of the conoid, the conoid fibers are cut obliquely as in Fig. 4 A. Tilting the section in the microscope brings the conoid fibers on one side of the conoid into an end-on view, whereas on the other side they are brought into a side view (Fig. 6, A and B). If the direction of tilt is such that the top of the conoid moves away from the viewer, then the conoid fibers on the left-hand side of the conoid become parallel to the direction of view (Fig. 6 A), whereas the right hand fibers become perpendicular to the view direction. Tilting in the opposite direction (Fig. 6 B, top towards viewer) reverses the appearance of the left and right sides. These observations establish that the conoid fibers follow a left-handed helical path.
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Rotationally and translationally aligned cross-sectional profiles of a number of conoid fibers were superimposed to obtain an average (Fig 7). In the best individual cross-sections, and in the average, several features can be seen that distinguish these fibers from typical singlet or doublet microtubules. Most obviously, the protofilaments of conoid fibers are not wrapped into a closed tube with circular cross-section. Nor do these protofilaments lie along an arc of a circle, i.e., these protofilaments assemble into a nonsymmetric structure, they are not simply incomplete microtubules missing a small sector. In addition to the nine protofilaments that are clearly outlined in negative contrast by the tannic acid staining protocol (Fig. 7 B, black ellipses), additional small structures with lower contrast are consistently seen (Fig. 7 B, gray "arms"). These are likely to represent microtubule-associated proteins (MAPs)* (or motor molecules) attached to the tubulin protofilaments, as suggested by Fourier analysis of images of frozen hydrated specimens (discussed below). We note that the sum of 14 fibers, 430 nm long, with nine protofilaments, plus the two 350-nm intraconoid microtubules closely matches the estimated tubulin content of the apical region (Swedlow et al., 2002).
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Longitudinal periodicities in individual conoid fibers (Fig. 8 B) are difficult to see by eye, but Fourier analysis of images from frozen hydrated samples reveals a rich set of periodic features. Power spectra were computed from individual conoid fibers that were digitally cut out of the image and digitally straightened. Summing the power spectra from several conoid fibers, clear 8- and 4-nm layer lines are visible (Fig. 9, top). This periodicity is characteristic of MAP-decorated microtubules (Amos and Klug, 1974; Amos, 1977; Kim et al., 1979; Murray, 1986a; Amos and Amos, 1991a). In a few cases, these two-layer lines could be seen in power spectra from individual conoid fibers. Remarkably, power spectra from conoid fiber images in glancing sections such as Fig. 4 B, whereas somewhat noisy, nevertheless showed the 8-nm longitudinal periodicity. The distribution of intensities along the equator of the power spectra (corresponding to the Fourier transform of the radial mass distribution of the object) is similar for subpellicular and intraconoid microtubules, but the outermost strong equatorial peak occurs at slightly smaller reciprocal space position for the conoid fibers than for the other two structures (5.4 nm vs. 5.0 nm). Weaker layer lines are also visible in the power spectra from some conoid fibers, suggesting that their true repeat length is 96 nm (Amos and Klug, 1974; Amos, 1977), as it is for the intraconoid and the subpellicular microtubules (Fig. 9, middle two spectra).
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Several different patterns of periodic longitudinal density modulation are seen in subpellicular microtubules (Fig. 8 C, arrows), depending on direction of view and extent of defocus of the objective lens. Some fragments show a faint beading along the straight longitudinal protofilament striations (Fig. 8 C, upper inset) as is typical of microtubules in other types of cells (Amos and Amos, 1991a; Wade and Chretien, 1993). Other fragments show strong 8-nm striations running across the microtubule, almost perpendicular to the long axis (Fig. 8 C, lower inset). In a few cases, we have found microtubules standing on end in these preparations (Fig. 8 E), a view that has not previously been reported for native microtubules. These end-on views may arise from intraconoid microtubules, which, in contrast to conoid fibers and subpellicular microtubules, are always perfectly straight.
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Discussion |
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T. gondii has one copy each of genes for - and ß-tubulin (Nagel and Boothroyd, 1988), which are utilized for several different macromolecular assemblies, including spindle, intraconoid and subpellicular microtubules, and conoid fibers. Surprisingly, although the conoid fibers are composed of the same tubulin isoforms and have several structural features in common with microtubules, they are not microtubules at all. In microtubules, columns of dimers (protofilaments) can be seen in cross sections if care is take to orient the microtubule exactly parallel to the direction of view. In this orientation, the microtubule looks perfectly circular, with distinct, countable protofilaments around its entire circumference (Figs. 6, D and E, and 8 E). Very slight misorientation (
5° in a typical EM section) blurs the images so that individual protofilaments can no longer be counted on two sides, but the profile remains circular; misorientation of
20° causes the profile to become elliptical.
Side views of conoid fibers (Figs. 4 B, 5 A, and 8 B) show longitudinal striations, suggesting that their subunits are arranged in straight columns similar to microtubule protofilaments. We confirmed this arrangement by showing that careful orientation does reveal distinct protofilaments in cross sections of the conoid (Figs. 6 and 7). However, unlike microtubules, the conoid fibers are not tubular and contain only 9 protofilaments. Protofilaments are more difficult to count in conoid fibers than in microtubules because perfect orientation is much harder to achieve (due to the curvature of the fibers themselves as they spiral around the conoid wall), but the cross-sectional profile of tubulin protofilaments is the same on both sides of the conoid shown in Fig. 6 (for example), confirming the proper orientation of this sample. Thus, it is clear that the observed deviation from circular symmetry inherent in the comma shape of conoid fibers cannot be an artifact of misorientation.
The lack of circular symmetry also indicates that the conoid fibers are not derived from ordinary microtubules simply through loss of a few protofilaments. Selective protofilament loss has been observed with heat or other treatments that partially depolymerize microtubules in other types of cells (Behnke and Forer, 1967; Witman et al., 1972; Tilney et al., 1973), but the remnant structures are always C- or S-shaped in cross-section; i.e., arcs of circles. Sheets of protofilaments that fail to form a closed tube can also be observed under certain polymerizing conditions in vitro, but these also consist of arcs of circles when viewed in cross section (Burton and Himes, 1978; Mandelkow and Mandelkow, 1979; Pierson et al., 1979; McIntosh and Euteneuer, 1984; Amos and Amos, 1991a; Mandelkow et al., 1991; Chretien et al., 1995). A transition between a closed tube form and a helical ribbon has been reported in Allogromia (Welnhofer and Travis, 1998), but that structure does not resemble the comma shape observed in conoid fibers. We conclude that the conoid fibers represent a previously unknown arrangement of tubulin.
Along the length of the conoid fibers, tubulin molecules are spaced 4 nm apart, just as in microtubules. It seems probable that conoid protofilaments exhibit parallel, as opposed to antiparallel, orientation (implying that the fiber as a whole is a polar structure) as has been observed in all naturally occurring tubulin polymers, but the signal-to-noise ratio in images of conoid fibers obtained to date is too low to confirm this supposition. However, the lack of circular symmetry in cross-section means that the lateral association of protofilaments in conoid fibers must be quite different from microtubules. The noncircular cross-sectional profile implies that the bonds between adjacent protofilaments are very different at one end of the comma compared to the other: adjacent protofilaments at the curved end must be rotated by 30° with respect to each other (similar to the 360/13 = 27.7 degrees in a canonical 13 protofilament microtubule), whereas adjacent protofilaments at the tail of the comma are only reoriented by
15°. Could this asymmetric arrangement be an accommodation to the extreme curvature of the fibers as they spiral around the 380-nm diameter conoid? At this point one can only speculate that the energy required to maintain this nonequivalence (Amos and Amos, 1991b) is provided by the binding energy of other proteins associated with the conoid fibers (CAPS). Weak layer lines in the power spectra computed from conoid fiber images hint that a periodic super-lattice with a long repeat is present, indexing on 96 nm (as is typical for MAPS; Amos and Klug, 1974; Amos, 1977; Kim et al., 1979; Murray, 1986a; Amos and Amos, 1991a). Monoclonal antibodies against unknown peptides associated with the conoid have been reported (Morrissette and Roos, 1998; Sasai et al., 1998), and it will be interesting to see if these proteins associate directly with the conoid fibers, and/or T. gondii microtubules, and whether they exhibit significant similarity to known microtubule binding proteins (Lewis et al., 1988; Noble et al., 1989; Hemphill et al., 1992; Doll et al., 1993; Perez et al., 1997).
FRAP measurements reveal rapid incorporation cytoplasmic tubulin into developing conoids during the early stages of daughter parasite assembly, but not into mature conoids. Similarly, the subpellicular microtubules of daughter parasites incorporate tubulin at their growing ends, but are not in equilibrium with the cytoplasmic pool after elongation has ceased. This change in dynamic properties is consistent with results from previous studies using drugs that affect microtubule stability (Stokkermans et al., 1996). Formation of subpellicular microtubules is blocked upon treatment with oryzalin (Shaw et al., 2000; Morrissette and Sibley, 2002), but preformed microtubules are stable in the presence of the drug. Neither taxol nor dinitroanilines affect preformed conoids (Shaw et al., 2000). The lower polar ring has been postulated to be the microtubule-organizing center for the subpellicular microtubules (Russell and Burns, 1984). Daughter conoids are apparently formed de novo in each cell cycle at some distance from the maternal conoid, implying that no template is needed for initiation of conoid fiber assembly.
The conoid undergoes a Ca2+-dependent translation of 0.5 µm relative to the lower polar ring (Mondragon and Frixione, 1996), causing it to extend at the apical end of the parasite. We have now observed a second form of motion correlated with extension, an increase in pitch of the conoid fibers. It is not clear whether extension and increased pitch result from independent motors responding to the same activation signal, or whether one motion is a simple mechanical consequence of the other. The nature of the motor(s) is unknown. The two intraconoid microtubules are in the right location to serve as tracks for a dynein/kinesin powered movement, but none has yet been reported in this location. Cytochalasin D, which blocks the Ca2+-dependent motile activity required for parasite exit from the host cell (Dobrowolski and Sibley, 1996, 1997), has been reported to partially block conoid protrusion in response to Ca2+ ionophore treatment (Mondragon and Frixione, 1996), suggesting involvement of a myosin. However, the maximum inhibition observed was only
50%. Another important unanswered question is how the matrix surrounding the conoid fibers participates in these movements. The new polymer expands our knowledge of tubulin assemblies, but also lengthens the list of questions to be answered.
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Materials and methods |
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A23187 treatment and detergent extraction
YFP--tubulin parasites (1 T25 flask) were harvested and washed, then incubated in 10 µl 20 µ M A23187 in PBS at room temperature for 1 h. For extraction, parasites were centrifuged and resuspended in 20 µl 10 mM deoxycholate in distilled water (pH 7.0). 23 µl extracted parasite suspension was put on a slide, covered with a coverslip, sealed with nail polish, and imaged immediately.
Immunofluorescence labeling
A 10-µl drop of parasites was allowed to adhere to a coverslip for 1 h in a hydrated chamber, fixed 10 min with 4% formaldehyde in PBS, extracted 40 min with 10 mM deoxycholate in Mg2+PBS, blocked with 3% BSA in PBS, and labeled with monoclonal anti-tubulin antibody (B5-1-2, diluted 1:1,000 in PBS + 3%BSA; Sigma-Aldrich) followed by Alexa594 goat antimouse IgG (1:1,000 diluted in PBS + 3% BSA; Molecular Probes).
Immunogold labeling
RH parasites were resuspended in 30 µl 10 mM deoxycholate and rotated at 20°C for 30 min. 3 µl of extracted parasite suspension was allowed to settle on a carbon coated nickel grid for 7 min in a humid chamber. Grids were washed 5 min with water, fixed 5 min with 4% formaldehyde in PBS, and then washed 3 x 5 min with PBS. Free aldehyde groups were blocked by incubation with 50 mM glycine in PBS for 20 min (pH 7.5) followed by 15 min incubation with 0.1% NaBH4 in PBS. Grids were washed twice with PBS, blocked with 5% BSA + 0.1% fish gelatin in PBS for 30 min, washed 2 x 5 min with incubation buffer (0.8% BSA and 0.1% fish gelatin in PBS, 15 mM NaN3), incubated with primary antibodies (a mixture of mouse anti- [B5-12; Sigma-Aldrich] and anti-ß tubulin [E7; Sigma-Aldrich] diluted 1:50 in incubation buffer with 15 mM NaN3) for 3 h, washed 6 x 5 min in incubation buffer, inverted on 10-µl drops of secondary antibody (goat antimouse IgG-conjugated with ultra-small gold (EMS Inc.) diluted 1:20 in incubation buffer), incubated overnight at 4°C, and then washed 6 x 5 min with incubation buffer and 2 x 5 min with PBS. The samples were postfixed 5 min with 1% glutaraldehyde in PBS, followed by 3 x 5 min wash in distilled water. Silver enhancement was carried out using the HQ silver enhancement kit (Nanoprobes Inc.) by floating grids on mixtures of the initiator, activator, and modulator for 45 s to 1 min in a light-tight chamber, and then washing 2 x 5 min with water. Grids were negatively stained using 2% phosphotungstic acid (pH 7.0).
Western blotting
Parasites were harvested, washed with PBS, lysed with 0.5% NP40, 2 mM EDTA in PBS, immediately put in SDS gel loading buffer, and heated at 70°C for 20 min. Samples were run on a 412% Bis-Tris SDS gradient gel (Invitrogen) at 200 V for 1 h and electroblotted onto nitrocellulose membranes. After incubating in blocking buffer (5% fat-free dry milk in PBS with 0.02% Tween-20), blots were labeled with monoclonal mouse anti-GFP (Clontech Laboratories, Inc.) or monoclonal mouse anti-tubulin (Sigma-Aldrich; B5-12) diluted 1:1,000 in blocking buffer. Bound antibody was detected with goat antimouse IgG conjugated to horseradish peroxidase (1:3,000 in blocking buffer; Bio-Rad Laboratories), using the Supersignal West Pico Kit (Pierce Chemical Co.) according to the manufacturer's protocol.
FRAP
Host cell monolayers were cultivated in T chambers (Bioptechs, Inc.) and infected with parasites. 10 mM Hepes (pH 7.0) was added to the medium immediately prior to imaging at 37°C, using a Bioptechs heated microscope stage and objective lens heater. FRAP analysis was performed on a Zeiss LSM510 confocal microscope using the 488-nm line of an Ar/Kr laser.
Cryoelectron microscopy
Parasites were isolated and treated with A23187 to induce conoid protrusion, followed by deoxycholate extraction. 12 µl extracted parasite suspension was placed on a grid covered with holey carbon film and plunged into rapidly stirred cryogen (liquid propane + 5% isopentane, maintained at liquid nitrogen temperature) as previously described (Murray and Ward, 1987a, 1987b; Ward and Murray, 1987). Frozen grids were searched with a Philips 400 EM at low illumination with a defocus of 0.8 mm to find appropriate regions for imaging. The EM was focused off axis at x220,000, and micrographs were acquired using the Philips low dose unit at x22,000, x28,000 or x36,000 magnification, with an electron dose of
500 e-/nm2, at 120 kev and defocus of 1.6 or 2.0 µm, on Kodak SO-163 film developed for maximum contrast.
Conventional EM of thin sections
2 T25 flasks of RH parasites were harvested, treated with A23187, and suspended in 2 ml 0.1 M NaMES, 2 mM MgCl2, 100 µM CaCl2, 1 mM CaEGTA, pH 6.6 (Buffer 1). Parasites were lysed by adding TX-100 to 0.5%, incubated 15 min, washed with Buffer 1, fixed in Buffer 1 plus 1% glutaraldehyde and 1% tannic acid for 6 h at 37°C, centrifuged, and resuspended in 1 ml 15 mM NaH2PO4 (pH 6.0) plus 1.3% OsO4 at 4°C for 1 h. After centrifugation, the pellet was rinsed with H2O, stained with 2% UrAc for 10 h, dehydrated, and embedded in Spurr resin. Dark grey sections (estimated to be 2530 nm thick) were cut and stained with saturated UrAc:MeOH (1:1) for 1.5 h, and then with lead citrate for 4 min. Sections were imaged at 120 kev in a Philips 400 electron microscope.
Image analysis
Micrographs were scanned on a Perkin-Elmer 1010G microdensitometer at a pixel spacing of 5, 10, or 25 µm on the film. Data was transferred to an Indigo Iris work station for analysis using the Semper image processing software (Synoptics Ltd.).
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Footnotes |
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Acknowledgments |
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This research was supported by National Institutes of Health grants R01 A149301 (J.M. Murray and D.S. Roos) and R21 GM60216 (J.M. Murray). D.S. Roos is a Burroughs-Wellcome Scholar in Molecular Parasitology.
Submitted: 18 December 2001
Revised: 1 February 2002
Accepted: 6 February 2002
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References |
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