Figure 1.
Trypanosome swimming behavior is a function of viscosity.
(A) Illustration of Trypanosoma brucei cell architecture. (B) Percentage of motile cells (squares) and mean population velocity (circles) in cell culture medium of varying viscosity, adjusted by the addition of methylcellulose (see Table 1 for concentrations; squares: n = 600–1.000 cells, weighted mean and weighted standard deviation from 3 independent experiments; circles: n>100 cells, mean and standard error). A cell was classified as motile if its trajectory exceeded a length of 150 µm, which corresponds to an average velocity of 5 µm s−1 during the 30 seconds of observation. (C, D) Trajectories of trypanosomes moving for 30 seconds in media with viscosities of 1 mPa s (C) and 5 mPa s (D). Trajectories showing less than 150 µm of directional motion are indicated in red and correspond to non-persistently swimming cells. Cells marked with an asterisk were in the final stage of cell division, having two opposing flagella and exhibited tumbling paths characteristic for this stage.
Table 1.
Viscosity of the medium affects motility of trypanosomes.
Figure 2.
Efficient trypanosome motility requires mechanical interaction with obstacles similar to red blood cells.
(A) Time-projection of a fluorescently labeled trypanosome (green) moving in a regular pillar array with 4 µm spacing. The fluorescence projection image was merged with a bright-field image of pillars, which had been transformed to a binary image and false-colored red. Within these 4 µm-pillar arrays cells reached their maximum swimming velocity of about 40 µm s−1. Inset: Illustration of the experimental setup. (B) The pillar spacing determines the percentage of persistently swimming cells. Almost 90% of trypanosomes were classified as motile cells in arrays with 4 µm pillar distance, which corresponds to the mean spacing of erythrocytes in blood (n = 150). Wider spacing led to a sharp decrease in directional motility. (C) The dynamics of the cells net directional movement as represented by the mean squared displacement (MSD) plotted over the observation time points to a fast, super-diffusive swimming behavior in pillar arrays of 4 µm spacing. Trypanosomes propagate far more effectively in 4 µm-spaced pillar arrays than in wider spaces. (n = 12; mean and SEM). (D) Maximum intensity projection (left) and selected still images from a high-speed fluorescence (xyt) image series of a fluorescently labeled trypanosome swimming through an array of pillars (4 µm spacing), acquired at a frame-rate of 400 images per second (Video S7). Note that the curvatures of the flagellum and the cell body closely reflect the shape of the pillars (positions indicated by ‘x’). Scale bar = 5 µm.
Table 2.
Maximum swimming velocity of trypanosomes in different media.
Figure 3.
Trypanosome mode of motility is a consequence of the architecture of the cell, which resembles an adaption to life in blood.
(A) Volume-rendered models of trypanosomes, surface-labeled with AMCA-sulfo-NHS. The course of the flagellum attached to the cell body (closed arrows) is clearly visible from the flagellar pocket (open arrows) to the anterior free end. The flagellum characteristically describes a turn of about 180 degrees around the cell body, counter-clockwise in swimming direction. This can unequivocally be seen in the animated three-dimensional view of the volume models (Videos S2, S3). The cells are representative examples of directionally swimming trypanosomes, as was confirmed by matching the 2D-views of the model with the single images from fluorescence high speed videos (Fig. 3B and Video S4). (B) Time-projection (xyt) of a directionally swimming cell fluorescently labeled with Atto488-NHS and recorded at 400 fps (Video S4). The projection reveals a wave pattern with a frequency of 2–3 Hz and amplitude of 3.6±0.2 µm. This amplitude matches the radius of erythrocytes as illustrated by dashed circles. The width of the swimming trypanosomes trajectory is close to 4 µm, which is not only optimal for efficient motion in pillar arrays (Fig. 2), but also corresponds to the mean distance of erythrocytes in blood. Note that no pillars were present in this experiment. Thus, the appearance of the characteristic swimming trail of trypanosomes is not a consequence of the presence of obstacles, but a product of cell shape and mode of motility and hence it is genetically fixed. (C) Out-of-focus information supports the view of a helical type of trypanosome movement. Top: False-colored time projection of a bright field image series, recorded at 500 fps (Video S8). The average intensity in the bright field images corresponds to the z-position of the respective parts of the cell body. In this case, the parts of the body that are out-of-focus while swimming through the field of view are imaged with brighter intensity. This information is quantified in the false colored xyt-projection. Following the trypanosomes path, high intensities (red) and lower intensities (green) periodically alternate, corresponding to out of focus and in focus regions respectively, as seen in the single images of the time series (below). Taken together, the directionally swimming cells describe characteristic wave patterns along all three spatial axes. Considering the observed pattern of flagellum movement relative to the cell body, these results strongly support a rotational motion of persistently swimming trypanosomes around their anterior-posterior axis, which represents an adaptation to life in blood.
Figure 4.
Time-dependent tomography proves the rotation of the cell body during locomotion.
(A) A bright-field image series of a directionally swimming trypanosome was acquired at 500 fps. Successive flagellar beats were analyzed and one image depicting the beginning of each beat was selected. This was every 28th frame, corresponding to a beat frequency of 18 Hz (left). After six beats the cell periodically reached the same spatial orientation. The edges of the cell were traced in the two-dimensional images and extruded to create three-dimensional objects. The six resulting 3D-contours were aligned on one anterior-posterior axis. The models were successively rotated around this axis in 60° steps per beat. The intersecting regions of the rotated objects were calculated and extracted. This produced a three-dimensional volume object that closely resembled a trypanosome cell body. When rotated by 60° per step the resulting views of the three-dimensional body compared well with the original microscopic images (right). The flagellum was traced and modeled manually, as the flexible, free anterior part cannot be aligned together with the cell. The flagellum (green) is shown superimposed on and rotated together with the cell body (right). This “time-dependent“ tomography method only produced a recognizable trypanosome model assuming 60°±5° turns per beat. All analyzed cells produced valid models in the range of 50°±10°. Note that these results formally prove a rotational mode of trypanosome motion. (B) Schematic illustration of the principle of time-dependent tomography.
Figure 5.
The plane-rotational motion of directionally swimming trypanosomes.
(A) Bright-field images from a xyt-series acquired with 500 fps (Video S8). Each image shows the beginning of successive up- or down-strokes of the flagellar tip, meaning a new wave starts at the anterior end of the flagellum every two images, i.e. after 28 frames. At each point in time, 2–3 wave crests are visible progressing from the anterior (tip of flagellum) towards the posterior part of the swimming cell. Every single up- and down-stroke causes forward motion in the opposite direction of wave propagation, seen as discrete translocations of about 0.7 µm, due to the helical path of the body and rotation of the cell body by about 25 degrees counter-clockwise. The cell was swimming at a speed of 25 µm s−1 and travelled 9.2 µm in 364 ms. In this period the flagellum produced 6.5 beats. (B) Illustration of the plane-rotational mode of motion compared to a bihelical mode (14). The latter requires a reversal of the rotational motion of the cell body after a turn of 180 degrees (marked by red asterisk), while the plane-rotational motion results in a continuous rotation in the same direction. Although the overall impression of these models appears rather similar, the physics underlying these two types of motion are fundamentally different.
Figure 6.
A triangulated surface model of an African trypanosome.
(A) The elongated cell body is modeled through a net of vertices connected by springs and an additional bending rigidity (for details see Materials and Methods section). (B) (a) Side view of the trypanosome model. (b) The dotted line shown in (a) discretized into vertices along which the bending potential is applied, (c) shows how the tangent vectors ti, ti+1 and the angle θ are defined using the discretized vertices. (C) The trypanosome surface model at the beginning of the simulation with the helical flagellum indicated as the blue colored line. (D) Average flow field of the model trypanosome swimming as obtained from simulation with multi-particle collision dynamics.
Figure 7.
The simulated motion of the trypanosome model using multi-particle collision dynamics confirms the experimentally observed mode of motion.
Panels 1–18 show selected frames of an exemplary simulation (see Video S13). The pictures illustrate four full cell rotations. The path of the posterior pole of the cell is shown in cyan color. Due to rotation of the cell body the simulated flagellum (blue color) always appears and disappears on opposite sides of the cell.