Structure of accounts for its bihelical motion

Alexey Y. Koyfmana, Michael F. Schmida, Ladan Gheiratmandb, Caroline J. Fua, Htet A. Khanta, Dandan Huangb, Cynthia Y. Heb, and Wah Chiua,1

aNational Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030; and bDepartment of Biological Science and Centre for BioImaging Sciences, National University of Singapore, Singapore 117543

Edited by Wolfgang P. Baumeister, Max-Planck-Institute of Biochemistry, Martinsried, Germany, and approved May 18, 2011 (received for review March 8, 2011)

Trypanosoma brucei is a parasitic protozoan that causes African sleeping sickness. It contains a flagellum required for locomotion and viability. In addition to a microtubular axoneme, the flagellum contains a crystalline paraflagellar rod (PFR) and connecting pro- teins. We show here, by cryoelectron tomography, the structure of the flagellum in three bending states. The PFR lattice in straight flagella repeats every 56 nm along the length of the axoneme, matching the spacing of the connecting . During flagellar bending, the PFR crystallographic unit lengths remain constant while the interaxial angles vary, similar to a jackscrew. The axo- Fig. 1. Trypanosoma brucei flagellum. (A) Diagram of a trypanosome cell neme drives the expansion and compression of the PFR lattice. (blue) with attached flagellum (yellow). (B) Slice of a tomographic reconstruc- We propose that the PFR modifies the in-plane axoneme motion tion of an isolated straight flagellum. The flagellum, composed of an axo- neme, a PFR (12), and connecting proteins, is attached to the cell body. to produce the characteristic trypanosome bihelical motility as captured by high-speed light microscope videography. PFR is demonstrated by the computed diffraction pattern of a

single image (Fig. S2), which shows diffraction spots expected BIOPHYSICS AND rypanosoma brucei

has devastated the African continent for for a crystal. In other tomograms, we have observed flagella in COMPUTATIONAL BIOLOGY Tcenturies by infecting humans and domestic animals and bent conformations, similar to those observed during flagellar has hindered economic development in sub-Saharan Africa motion. Because these three flagellar components (PFR, axo- (1). Current sleeping sickness treatments are inadequate and neme, and connecting proteins) exhibit different periodicity and the drugs used are highly toxic (2). In recent years, the motility symmetry, we had to use different strategies to align and average of the T. brucei flagellum has been found to be essential for para- each of them separately (Materials and Methods). The PFR has site survival, infection, and disease pathogenesis (3), and has been found to contain multiple regions (24). We observe these emerged as a promising drug target (4). Flagella with similar distinct regions of the PFR in unaveraged, raw tomogram cross- structural organization and composition have also been sections (Fig. S3C and Movie S3). However, in our method of found in euglenoids (5) and other kinetoplastid parasites includ- alignment, the crystallinity of the largest and most well-ordered ing Leishmania spp. and Trypanosoma cruzi, which cause Leish- portion of the PFR (the distal portion) strongly influences the maniasis and Chagas disease, respectively (6). overall PFR average. The proximal PFR region was not observed The trypanosome flagellum is more complex than most other after the averaging because it does not possess such crystallinity. eukaryotic -based flagella (7–9) and is completely The final averaged structure of the entire flagellum (Fig. 2A different from rotary-motor based bacterial flagella (10). Each and Movie S3) was reconstituted from the averaged components T. brucei cell contains one flagellum that moves the cell body of straight flagella. in an alternating right and left-handed twist resulting in bihelical The averaged PFR density was derived from five different motion (11) (Movie S1). The membrane-enclosed flagellum, tomograms (Fig. 2 A and B). The PFR is a three-dimensional pro- composed of an axoneme, a paraflagellar rod (PFR) (12), and tein lattice that has crisscrossing densities and a large proportion connecting proteins, is attached to the cell body (Fig. 1). PFR of empty volume, both of which are reminiscent of the crystal was identified as a lattice-like ultrastructure in T. brucei flagellum structure of tropomyosin (25, 26). The linear densities are paral- (13). This periodic and crystalline nature of the PFR was con- lel to the crystallographic unit cell axes and may correspond to firmed in T. brucei (14) and related species (15, 16). Monoclonal one or more parallel coiled-coil bundles of the major PFR pro- antibody screens (17) and proteomics studies (18–20) have iden- teins. The diagonal of the crystallographic PFR unit cell repeats tified at least 40 PFR proteins. Among them, PFR1 (73 kDa) and at 56-nm intervals in the direction parallel to the axoneme PFR2 (69 kDa), containing coiled-coil regions (21), are major (Fig. 2B). We used a skeletonization algorithm (27) to provide structural components of the PFR (22). Depletion of these pro- a simplified representation of the densities in the PFR lattice teins results in failure of PFR assembly and cell motility defects (17, 23) (Fig. S1 and Movie S2). In the T. brucei pathogenic blood- Author contributions: A.Y.K., M.F.S., L.G., C.J.F., H.A.K., D.H., C.Y.H., and W.C. designed stream form, ablation of PFR2 causes death of the parasite (18). research; A.Y.K., L.G., C.J.F., H.A.K., and D.H. performed research; A.Y.K., M.F.S., C.Y.H., These results demonstrate a critical role of the PFR in T. brucei and W.C. analyzed data; and A.Y.K., M.F.S., C.Y.H., and W.C. wrote the paper. motility and viability. We have employed cryoelectron tomogra- The authors declare no conflict of interest. phy (cryo-ET) to determine the structure of a biochemically This article is a PNAS Direct Submission. T. brucei isolated flagella (18). We describe here a model that Freely available online through the PNAS open access option. explains how the structure and arrangement of the flagellar Data deposition: The 3D cryoelectron tomographic averages have been deposited in components produces the bihelical motion of the flagellum. the Electron Microscopy Data Bank, www.emdatabank.org (accession nos. EMDB-5302, Fig. 1B is a projection through 30 slices (33 nm) of a tomogram 5303, 5304, 5305, and 5306). of the T. brucei flagellum showing three components of a straight 1To whom correspondence should be addressed. E-mail: [email protected]. flagellum: the crystalline PFR, the axoneme, and the proteins This article contains supporting information online at www.pnas.org/lookup/suppl/ connecting the PFR to the axoneme. The crystallinity of the doi:10.1073/pnas.1103634108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1103634108 PNAS Early Edition ∣ 1of4 Downloaded by guest on September 25, 2021 similar to axonemes from other (7, 8, 37–40). How- ever, structures such as the outer arms could have been partially removed during the extraction with 1 M KCl. Two rows of connecting proteins (red and pink in Fig. 2 A–C) between the PFR and the axoneme were identified. We observe substantial connections to doublets 5 and 6, but not to 4 and 7 as previously visualized and reported (3, 41). The connections to doublets 4 and 7 are not as bulky as those to doublets 5 and 6. They are longer and perhaps more flexible, and therefore not readily visible in the raw tomograms. Indeed it was shown that connections to doublets 4 and 7 are thin linear structures (13, 14, 16, 24, 42). Due to the difficulty in visualizing the con- nectors to doublets 4 and 7, they were not chosen for averaging from our reconstructions. The connecting proteins which we do observe (to doublets 5 and 6) repeat every 56 nm in straight flagella, a distance corresponding to seven tubulin dimers along the axoneme (Fig. 2B). This distance is the same as the PFR repeat along the axoneme and also similar to periodic attach- ments seen in the Euglena axoneme (5). Each of the two rows of connecting proteins was averaged along the length of the flagella. The averages of the connecting proteins in the two rows are similar in size (Fig. 2B and Movie S3) and are schematically represented by spheres in Fig. 2C. It is likely that each connecting density is a complex of several proteins. Fig. 2. T. brucei flagellum components. (A) Flagellum cross-section. The ax- Whereas straight flagella were used to determine the average oneme is radially colored: central pair complex (yellow), radial spokes (light structure, bent flagella can suggest a model for their motion. Fig. 3 green), microtubule doublets (blue). The dashed red line (bisecting the cen- A–F tral pair) represents the plane of bending of an axoneme. The PFR is offset show top and side views of tomogram slices of the PFR from from that plane (dashed purple line through the middle of the PFR). (B) The three differently bent flagella. The PFR averages are shown in diagonal (shown by a horizontal white arrow) of the crystallographic unit cell different colors. The crystallographic unit cell lengths of bent of the PFR (green) repeats with the same spacing (56 nm) as that of the and straight PFR agree within 5%: a ¼ 52 2, b ¼ 46 2, and connecting proteins (red and pink) along the axoneme axis. (C) The layered c ¼ 22 1 nm (a total of 7,000 unit cells went into these nature of the PFR. Skeletonized (27) PFR density is shown in gray, the crystal- averages) (Fig. 3G, Fig. S5, and Movie S3). Because the linear lographic unit cells are shown as green parallelograms, the connecting PFR protein densities are parallel to the crystallographic unit cell proteins are represented as spheres, and the are cylinders. axes rather than to the axoneme axis, the PFR proteins are not (D) T. brucei axoneme internal components. compressed or bent (Fig. 3) when the axoneme bends. Rather, the (Fig. 2C). A green parallelogram, passing through the highest PFR densities pivot as if on hinges at the corners of the crystal- PFR densities, represents four crystallographic unit cells in a plane lographic unit cell with a scissor-like motion, resulting in interax- (Fig. S4). Fig. 2C shows the layered nature of the PFR and its ial angular variations from 85° to 112° (Fig. 3 G and H). spatial relationship to the axoneme (Movie S3). The distance be- The organization and structural flexibility of the individual tween adjacent layers corresponds to the c crystallographic unit flagellar components and their relationship to each other suggest cell spacing (22 nm). Note that none of the crystallographic a mechanism for the movement of the trypanosome powered by axes is parallel or perpendicular to the axoneme axis. the beating axoneme. It is well accepted that the axoneme powers The axoneme average (Fig. 2D and Movie S3) was derived flagellar movement (32, 43, 44). Bending of the axoneme makes from a single tomogram that had the best-preserved structure. the connecting proteins bend along with it, causing their spacing It contains the characteristic nine outer microtubule doublets to shorten or lengthen from the 56-nm spacing in straight flagella (28–30) (blue) arranged around the central microtubule pair (Figs. 3H and 4A). This change in spacing of the connecting pro- (yellow) similar to those found in sea urchin (8). All nine teins in turn compresses or stretches the PFR along the axoneme microtubule doublets, with their associated structures, were direction (Fig. 3). Our observations suggest that each crystallo- aligned and averaged assuming that they were structurally equiva- graphic PFR unit cell is analogous to an automobile jackscrew lent, thereby compensating for the distortions caused by the (Fig. 3H and Movie S4). The crystallographic unit cell axes, cor- limited range of tilt angles (Materials and Methods). In our anno- responding to the rigid arms of the jackscrew and composed of the tation, microtubule doublet 1 (Fig. 2A and Movie S3) is distal coiled-coil structural proteins, remain constant in length, whereas to the PFR (31) and radial spokes (light green) connect outer the interaxial angles vary. The connecting proteins stretch and microtubule doublets to the central pair. Typically, axonemes compress the PFR hinges and thus correspond to the screw in bend in a plane bisecting the two microtubules of the central pair a jackscrew (Figs. 3H and 4, and Movie S4). The change in the (32) with a frequency of 10–20 Hz (33). Here, cryo-ET provides a crystallographic unit cell angles is analogous to the results of direct observation, in the same frozen specimen, of this orthogo- the action of the screw in a jackscrew. The coordinated action nal relationship between the central pair and the bending plane of the unit cells, illustrated by the parallelograms in Fig. 3G, pro- (Fig. S3 A and B). In our tomograms, the plane bisecting the two duce an overall stretching and contraction of the PFR in a manner microtubules of the central pair (Fig. 2A, red dashed line) and the analogous to an expansion gate (Fig. S6). Interestingly, using a plane through the connecting proteins (Fig. 2A, purple dashed flexible protein crystal as a part of a cellular mechanical device line) intersect at an angle consistent with previous measurements has been observed before (45). The actin-based acrosomal bun- (34–36). To statistically validate our observation, we measured dle, proposed as a biological spring (46), is another example of an the angle that the PFR makes with the perpendicular bisector of intracellular 3D protein crystal serving a biomechanical function. the central pair for a total of eight different flagella (Fig. S3 B–H, The preferred bending plane of the axoneme (34) makes an in addition to the tomogram for Fig. 2). We found it to be 20 7°. approximately 20° angle with the PFR (the angle between the Some features of the axoneme (Fig. 2D), such as the nine doub- dashed lines in Fig. 2A and Movie S3). A constraint on the bend- lets, the radial spokes, and the central microtubule pair, appear ing of the axoneme, like that imposed by the PFR having this 20°

2of4 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1103634108 Koyfman et al. Downloaded by guest on September 25, 2021 Fig. 3. The T. brucei flagellum acts like a biological jackscrew. Side views (A, C, and E) and top views (B, D, and F) of a tomogram of a PFR which is bent inward (A and B), straight (C and D), and bent outward (E and F). Tomogram slices are shown in gray. Aligned and averaged subtomograms are shown in color. (Scale bars: 100 nm.) (G) Top view of the crystallographic unit cell axes. While the axial distances stay constant, the γ angle changes as the PFR bends. (H) Jackscrew models of the PFR representing the 85° angle of the inwardly bent (expanded) crystallographic unit cell; 106° angle of the straight crystallographic unit cell; 112° angle of the crystallographic unit cell bent outward (compressed). Connecting proteins are represented by pink spheres.

offset, would induce a rotation, twisting the flagellum into a right- The trypanosome flagellum is the most complex microtubule- or left-handed helix (Fig. 4B and Movie S5). Fig. S7 shows this based nanomachine that has been studied by cryo-ET. Our obser- twist measured geometrically along a slightly bent flagellum in a vations support a molecular mechanism by which the properties single projection image. Fig. S8 shows the twist measured in 3D of the paraaxonemal structures shape the motion of T. brucei.We along the axoneme axis from a tomogram. Both methods give a propose a straightforward model for energy transduction, from twist of about 15° per 800 nm in slightly bent flagella. It is possible the beating axoneme through the connecting proteins to the that flagella were bent passively during freezing. However, no deformable PFR crystal, resulting in efficient cell propulsion of matter what caused these flagella to bend, we observe a consistent this trypanosome. relationship of bend to twist (Figs. S7 and S8). It is thus likely that Materials and Methods the isolated flagella are constrained to bend in the same way they BIOPHYSICS AND Isolation of T. brucei flagella was performed as previously described (18, 47). would in the motile trypanosome. We have observed long, 2 × 109 COMPUTATIONAL BIOLOGY μ Approximately procyclic 29.13 cells (48) were extracted sequentially straight flagella over a distance of up to 15 m. On the other with buffers containing 1% Nonidet P-40 and 1 M KCl. Isolated flagella hand, bent flagella occur only over short distances (less than were stored at 4 °C. Quantifoil Copper 200 mesh R 2∕2 grids were washed 1 μm) on the grid, possibly because the combination of bend overnight with ethyl acetate. Grids were pretreated with 15-nm gold nano- and twist would eventually cause them to come out of the plane particles for tomogram alignment. To prepare a frozen, hydrated grid, 2.5 μL of the thin ice layer. Movies S1 and S5 include high-speed light of flagella sample was applied to the grid, blotted, and plunged into microscopy showing the bihelical motion of the T. brucei flagella liquid ethane using a Vitrobot (FEI). The optimal ice thickness is around 19 3 300–400 nm, slightly larger than the diameter of the flagellum, as observed with a frequency of Hz (11). In contrast, the sea urchin in our tomograms. Imaging was performed on 200-kV microscopes sperm axoneme bends up and down with a simple sinusoidal mo- JEM2200FS and JEM2100 equipped with Gatan 4;096 × 4;096 pixel CCD cam- tion with a similar frequency (32, 33) (Movie S5). At the end of eras. The JEM2200FS has a field emission gun and an in-column energy filter, each half-cycle of the T. brucei axoneme beat, the direction of the whereas the JEM2100 has a LaB6 gun. Depending on the microscope and ima- twist reverses, generating a helix of the opposite hand (Fig. 4B ging conditions, the effective magnifications varied between 11,100× and and Movies S1 and S5) (11). Thus, the normal planar motion 16,500×. Tilt series were collected using SerialEM (49) targeted at 6–10 μm underfocus. Each 120° tilt series contained 60 images. Electron dose per of an axoneme is transformed into the observed bihelical wave 74 ∕ 2 movement characteristic of T. brucei. Depletion of the PFR tomogram ranged from 60 to electrons Å as typically used for cryo-ET T. brucei (50). Tomograms were reconstructed using IMOD (51). Subvolumes enclosing assembly (Fig. S1) of the flagella by RNAi (17, 23) segments of the PFR, the connecting proteins, and the axoneme were changes the bihelical motion into sinusoidal planar motion with extracted from the reconstructed tomograms. Initial models for the PFR a frequency of 18 1 Hz (Movie S2). The RNAi experiments re- and for the connecting proteins were created by aligning and averaging inforce the critical role of the PFR for the bihelical wave motion two adjacent subvolumes (52). More distal segments were then aligned to of the . Whereas other structures, including the cell and averaged with the initial model iteratively. A final realignment of each body, may influence the bihelical motion, the relative orientation subvolume to the model was performed to create a final average. The PFR of the PFR to the axoneme was observed to be consistent, unlike from bent flagella, recognized by visual inspection, were similarly aligned to each other. For the axoneme, 288-nm segments, representing three 96-nm the relationship of flagellum to the cell body (34). repeat lengths, were extracted along each microtubule doublet. The seg- ments overlapped each other by 96 nm. These segments were first aligned along their length by cross-correlation. The aligned segments were then aver- aged, after which the nine averaged doublets were then rotationally and translationally aligned and averaged with each other, mitigating the effect of the missing wedge. Finally, the original axoneme was reassembled by properly rotating and translating the microtubule doublet average back into the original volume in the nine original orientations. The separately aver- aged subtomograms of the PFR, of the connecting proteins, and of the axoneme were fitted back into an original tomogram density map. Density visualization was performed using Chimera (53).

ACKNOWLEDGMENTS. We thank Matthew Dougherty for help in preparing Fig. 4. Transformation of the normal axoneme (purple) planar motion into the movies, and Xiangan Liu and Ryan Rochat for help with illustrations. This the observed bihelical wave motion of T. brucei.(A) The relative sliding mo- research has been supported by National Institutes of Health through the tion of the microtubule doublets will tend to compress and expand the con- National Center for Research Resources (P41RR002250) and the Postdoctoral necting proteins (pink) (X1 >X2 >X3). (B) The presence of the PFR (green) Training Grant of National Institute of Allergy and Infectious Diseases that is offset 20° resists this tendency, leading to a bihelical twist of the (T32 AI07471 to A.Y.K.), and C.Y.H. is a research fellow of Singapore National attached flagellum and T. brucei cell. Research Foundation.

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