How does trypanosoma gambiense move

The time trajectory of each parasite was decomposed into distance, speed, and rotational angle to facilitate a more detailed quantitative vectorial analysis, and a population-wide analysis was also performed.

Epimastigotes from the T. For all experiments, the parasites were grown in homogeneous conditions and were sampled for use in experiments while in the logarithmic growth stage at 48 hours.

For motility assays, the movement of the labeled cells was measured and analyzed using the Image-Pro plus V 6. For this calculation, we used the following well-known formula from vector analysis, which we implemented in MATLAB 7.

The motion of free-swimming epimastigotes was analyzed using video microscopy, and the videos were processed with the Image-Pro plus V 6. Figure 1 illustrates the cell motility and flagellar beating of T. The parasites were highly motile, and showed great variability of movements and trajectories, which was characterized by alternating periods of translational cell movement, tumble and shutdown Figures 1 a , 1 b , and 1 c that resulted in different distance ranges traveled by the cells Figure 1 d.

The main waveform was a tractile beat that initiated at the tip of the flagellum and propagated toward the base, resulting in forward cell propulsion. In the free-swimming epimastigotes in which cell motion was not impeded, the wave amplitude did not change detectably along the entire free flagellum Figure 2 , as observed in Leishmania major [ 22 ].

This tractile beat was occasionally interrupted for short periods of base-to-tip wave beats that led to the reorientation of the anterior flagellar end and then the parasite body with no evident backward motility Figure 1 e light blue region, Figure 3 and Video 1. These data suggest that T. Even though the differences in the average velocities may have resulted from differences between cells in motion and cells attached to the microscope slide, all of the parasites had a vigorous motion, and the amplitude and frequency of the flagellar wave was constant.

The time trajectories of free-swimming epimastigotes through the liquid medium were decomposed into distance, speed, and rotational angle to facilitate a more detailed quantitative comparison. Vector analysis was then applied to characterize the speed and rotational angles of all individual trajectories of each parasite using a custom MATLAB 7. The speed data showed that the speed of the parasites could be described by a Maxwell-Boltzmann distribution, suggesting that epimastigotes undergo Brownian-like motion.

This finding indicated that there is no preference in the direction toward which parasites rotate, and it suggests a high degree of stochasticity. As the rotational angle histograms were not flat but rather showed a different bias for each parasite, the movement of parasites was not completely random, and the phenomenon was highly dependent on parasite behavior.

The smaller angles were produced during directional drift through directed motion, whereas the larger angles were produced during the spin and tumble stage when the flagellum does not show coherent directed motion. The rotational angle histogram for free-swimming parasite motion showed a peak centered at small angles, indicative of almost rectilinear paths, with few abrupt changes in direction.

This behavior was clearly observed when speed and rotational angle were analyzed together Figure 7. Taken together, these results indicate that epimastigotes from the same population taken from the same cell culture and exposed to identical environmental conditions do not follow a single motility mode. Even though that the motility process of trypanosomatids is important for survival, completion of the life cycle, and establishment of parasite infection, the molecular mechanisms involved remain poorly understood.

The recent publication of complete genome sequences for T. However, little is currently known about the general characteristics of motility and the flagellum beating processes, and an in-depth quantitative description against which hypotheses could be tested is lacking.

Using video microscopy and vectorial analysis of parasite trajectories, we have found that the motility of free-swimming epimastigotes of T. As for other trypanosomatids, the epimastigotes of T. The parasite motility analysis presented in this work indicates that the cell traces have patterns similar to those previously reported for T. This property of trypanosomatid flagella is distinctive compared with most other organisms in which beating is initiated from the proximal end of the flagella. In these organisms, it was believed that bend formation and sustained regular oscillation depended upon a localized resistance to interdoublet sliding, which is usually conferred by structures at the flagellar base, known as the basal body.

Similarly, in trypanosomes, it has been proposed that they may have some tip initiating or capping structure to provide resistance to doublet sliding in a manner similar to the basal body. This attachment structure has been found in Crithidia deanei , Herpetomonas megaseliae , Trypanosoma brucei , and Leishmania major [ 28 ]; therefore, this structure may also be present in T. The beat initiation from points other than the flagellum tip, as reported for L.

However, it was possible to detect that in addition to the tip-to-base flagellar wave, the epimastigotes also showed another type of beat that was propagated in the opposite direction base-to-tip, which is characteristic of ciliary beating at a much smaller frequency and in a highly asymmetric mode.

This ciliary beat started spontaneously and interrupted the initiation of the main tip-to-base beat, producing a sudden pause in which almost no movement was observed. Then, a short interval of base-to-tip waves was noted before the parasite reinitiated the tip-to-base flagellar beat that resulted in a very little translational motion and clearly produced a change in epimastigote orientation and the resumption of forward cell motion. This switch between flagellar and ciliary beating has been reported in T.

These results indicate that these organisms are able to sustain at least two kinds of beat types and suggest that they are likely able to maintain other types of beats when are in different microenvironments during their life cycle. The variability of movements and trajectories of T. These motility modes are the result of the cell elongation that correlates with cell stiffness, which has been shown to affect not only flagellar velocities but also the directionality of parasite movement [ 21 ].

Therefore, the predominance of directional motility in T. A recent analysis of the structural organization of the PFR of T. The PFR is composed of discrete filaments structured in lattice-like arrays with three distinct domains proximal, intermediate, and distal , and it has been proposed that the three domains may work together.

According to this proposal, when the proximal domain is compressed, the distal or opposite domain is stretched in an alternating pattern during flagellar beating. The intermediate domain would follow this dynamic of movement, and in a bent state, the filaments would get closer [ 5 ]. Therefore, the different motility modes observed during the swimming of epimastigotes could be an indication that the intermediate domain is able to change its position between the proximal and distal filaments, allowing the switch between flagellar and ciliary beating.

The movement of this domain would alternate the periods of translational cell movement, tumble, and shutdown of the parasite and result in the consequent reorientation of the epimastigotes. The mechanism by which the different modes of motility are coordinated is still unknown, but the minor components of the PFR [ 30 ] could participate as motor, anchoring, or connector proteins in the different modes of movement.

However, future biochemical and genetic studies will be necessary to determine the type, function, and possible participation in the flagellar beat of these proteins. The symmetry and equal amplitude waves through the flagellum observed in the directional motility of T. Quantitative analysis of the rotational angle and study of separated parasite displacement and flagellar beating, using the corresponding fixed and superimposed images, revealed that, as in Leishmania , T.

In contrast, in T. To test this hypothesis, future experiments comparing the flagellar beating of epimastigotes versus bloodstream and metacyclic trypomastigotes of T. The trypanosomatid flagellum is completely different from rotary-motor based bacterial flagella [ 32 ] and more complex than most other microtubule-based eukaryotic flagella [ 33 , 34 ].

Therefore, the quantitative description of motility and flagellar beating of T. In summary, our quantitative motility analysis results offer insights into flagellar beating of the American trypanosome and provide new detail on an important, yet poorly understood, motility mode of T. Manning-Cela, Grant no. Video 1. Video clips of CFSE green fluorescent labeled free-swimming epimastigotes analyzed by confocal microscopy. Ballesteros-Rodea et al. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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A Erratum for this article has been published. Ballesteros-Rodea, 1,2,3 M. Academic Editor: Abhay R. Received 15 Jul Revised 28 Sep Accepted 29 Sep Published 11 Jan Abstract The hemoflagellate Trypanosoma cruzi is the causative agent of American trypanosomiasis.

Introduction Trypanosoma cruzi is a flagellated protozoan that is the causative agent of Chagas disease, a debilitating and incurable fatal illness that affects 20 million people in Latin America [ 1 ]. The flagella are linked via a flagella connector-like structure as demonstrated by a single image of transmission electron microscopy TEM [ 10 ]. However, these approaches faced some limitations: SEM shows the cell topography but the kinetoplast and the nucleus are not visible hence preventing morphotype identification.

Furthermore, the new flagellum is detected only when it is visible outside of the flagellar pocket. In the case of TEM, single thin sections rarely show the relative position of the nucleus and the kinetoplast, as well as the presence of the mature and new flagellum. To circumvent these issues, we revisited T.

Here we show that the assembly of the new flagellum is initiated earlier than previously reported in PV trypanosomes committed to the asymmetric division.

Formation of a transition zone and elongation of the flagellum can already be detected in trypomastigote cells. In contrast to procyclic trypanosomes, flagellum construction is characterized by an early basal body segregation and the rapid acquisition of an independent flagellar pocket, followed by a late flagellum rotation when compared with procyclic cells. The flagella connector structure that links the new flagellum to the mature flagellum is present from the early stages of flagellum construction.

The AnTat 1. Flies were starved for at least 24 h before dissection performed at 14 or 21 days after infection. Tsetse were scrutinized under a MFC stereo microscope Leica, Nanterre, France for fluorescent parasites emitted by the TdTomato protein that is visible through the tsetse fly cuticle [ 32 ].

A total of 33 flies were dissected and the whole alimentary tract removed and placed on a glass slide containing a drop of PBS for immunofluorescence or in cold cacodylate buffer for electron microscopy EM experiments. The PVs were dilacerated using two 26 gauge needles to release trypanosomes.

Cells were left for 5 min to settle before fixation in cold methanol for 10 s followed by a rehydration step in PBS for 15 min. Image acquisition was performed using Micromanager software. For EM sample preparation, the entire digestive tracts of 11 flies of 21 days post-infection were placed in a drop of 0.

Entire proventriculi were then separated from the digestive tract and transferred to 1. Fixed samples were washed three times by the addition of fresh 0. The milling conditions for the trench that allowed the view of the cross-section were 10 nA at accelerating voltage of 30 kV. The fine polishing of the surface block was performed with 5 nA at 30 kV.

For the slice series, 1 nA milling current was applied removing a 10 nm layer from the specimen block surface. The voxel size was 10 nm in x, y, and z. Alignment of stacks was performed with the open source software ImageJ National Institutes of Health [ 38 ] and the Amira software was used for visualization v6. Segmentation and 3D reconstructions were performed manually using Amira software and a color code was attributed as follows: the new flagellum in orange; the mature flagellum in red; the kinetoplast in purple; and the nucleus in blue.

The new flagellum of trypanosomes was measured by segmenting the first slice of the basal plate up to the tip of the flagellum including the flagellar membrane. This is the first morphological modification related to the cell progression in the differentiation process, followed by elongation [ 10 ] Fig.

Figure 1 a shows a trypanosome containing an oval nucleus and a single flagellum with one TZ magenta and one axoneme green. A trypomastigote containing an elongated nucleus and a flagellum with one TZ and one axoneme is shown in Fig. A second fluorescent signal for FTZC was also observed in trypomastigotes with elongated nucleus Fig. The increase in distance between the two signals for FTZC and a more posterior position of the new flagellum in relation to the mature flagellum is presumably due to migration of the basal body of the new flagellum Fig.

The length of the mAb25 signal increased in trypanosomes where the nucleus was migrating towards the posterior end during the differentiation process from trypomastigote to epimastigote Fig. Flagellum assembly is an early event during T. A total of parasites were analysed, the percentages were calculated for trypomastigotes and transition forms. Flies were dissected 14 days post-infection. Abbreviations : Axo, axoneme; TZ, transition zone.

Note : Anterior and posterior regions of the cell are indicated on panel a. These results suggest that a new TZ and a new axoneme are already assembled in a significant proportion of cells displaying the trypomastigote morphology in the PV. Nevertheless, the signal intensity presumably associated to the new flagellum turned out to be weaker compared with the mature flagellum Fig.

Considering the differences in intensity of the fluorescent signals, it was crucial to verify the ultrastructural organization by electron microscopy techniques. The ion beam promotes micro abrasions on the sample surface exposing a fresh new layer and the electron beam scans over the block surface generating the image. This process is repeated for several micrometers and the collected images generate 3D stacks with a 10 nm resolution in Z-axis.

Two stacks of Entire PVs were immediately fixed after dissection maintaining the location of trypanosomes in their microenvironment and avoiding any contamination with parasites from the midgut and foregut. The processed PVs were oriented along longitudinal or transversal axes during embedding for better accessibility to the lumen where higher concentrations of trypanosomes were found.

The assembly of the new flagellum was investigated in these cells. The earliest stage of flagellum duplication was observed in trypomastigotes with an elongated nucleus Fig. Figure 2 a shows the ultrastructural organization of a trypomastigote cell containing a short new flagellum closely associated to the mature flagellum.

New and mature flagella were segmented including the flagellar membrane. This parasite was selected for 3D reconstruction Fig. The new flagellum orange is closely located to the kinetoplast purple Fig. The basal bodies are close to each other and separated by only nm. The bottom view of the cell allows the visualization of the entire elongated nucleus blue that is 6.

The most posterior end of the nucleus is separated from the kinetoplast center by 1. Figure 2 d shows the top surface view of the cell body and confirms that only the mature flagellum red emerges from the flagellar pocket Fig. The short new flagellum is inside the flagellar pocket at an early stage of elongation. The full stack is shown at Additional file 1 : Video S1.

The new flagellum is located in a more posterior region in relation to the mature flagellum. The diameter of the flagellum looks larger than that of the basal body because the membrane was used for segmentation. The cell body is in grey and the mature flagellum is in red. Notes : White and black arrowheads indicate the new and the mature basal bodies, respectively.

Anterior and posterior regions of the cell are indicated on panel a. Figure 3 a shows a section of a trypomastigote cell with the basal body Fig. The basal bodies are separated by nm. The axoneme reaches 1. The nucleus is closer to the kinetoplast, as they are separated by only nm Fig. The new flagellum emerges from its flagellar pocket during assembly.

The full stack is shown at Additional file 2 : Video S2. The new flagellum is closely associated to the kinetoplast and is in a posterior position in relation with the mature flagellum.

Anterior region and posterior regions of the cell are indicated on panel a. For transition forms, parasites were subdivided into early transition form Fig. In an early transition form, the kinetoplast is enlarged Fig. The basal body of the new flagellum is located close to the posterior tip of the kinetoplast Fig. The nucleus and the kinetoplast are occupying the same plane reflecting the moment when the nucleus reaches the posterior tip of the kinetoplast Fig.

This is accompanied by an obvious nucleus deformation Fig. Figure 4 d shows the top view of the cell with the new flagellum positioned to the left side relative to the antero-posterior axis of the cell body. A trypanosome in a late transition form is shown in Fig. The slice view shows the kinetoplast, the nucleus, the new flagellum and the mature flagellum Fig.

The nucleus migrates towards the posterior region and the kinetoplast is positioned at the anterior half of the nucleus region Fig. The basal body of the new flagellum Fig. However, the new flagellum is laterally positioned to the right side of the antero-posterior axis of the cell body.

This is in contrast with all previous stages where the new flagellum is positioned on the left side when looked from the anterior to posterior region of the cell body. The basal bodies are separated by 2. Transition forms where the nucleus migrates towards the posterior region of the body. Anterior region and posterior region of the cell are indicated on panel a and e. Flagellum elongation in proventricular trypanosomes was measured in cells corresponding to the stages described above Fig.

The new flagellum elongates progressively, consistent with the order suggested by IFA experiments. When this flagellum grows further and can be detected outside of the flagellar pocket in trypomastigote cells, its whole length is 1.

Flagellum elongation in parasites during differentiation in the proventriculus. Values are given as the mean and the standard errors SE are indicated. The numbers of parasites are shown in parentheses. The tip of the new flagellum was not visible in 8 cells, but in the remaining 71 cells an electron-dense structure was observed between the tip of the new flagellum and the side of the mature flagellum. This electron-dense structure seems to connect the new flagellum and the mature flagellum, similar to the flagella connector observed in procyclics [ 18 , 19 , 21 ].

Therefore, we carried out a TEM analysis of serial sections, an approach that provides more resolution for structural investigations. Three 80 nm-thick serial sections of a trypomastigote cell with two flagella are shown at Fig. In this cell, a tiny new flagellum is located inside the flagellar pocket, which is shared with the mature flagellum Fig.

This is the earliest stage of flagellum duplication in a trypomastigote cell that could be observed by TEM. The new flagellum consists of a TZ, a basal plate, and a short axoneme Fig.

A structure linking the new flagellum and the mature flagellum is present and exhibits an electron-dense trilaminate morphology connecting laterally the distal region of the new flagellum with the mature flagellum Fig. Starting from the base of the new axoneme, a first electron-dense plate is facing the basal plate of the new flagellum and the TZ region of the mature flagellum Fig.

The second plate is facing the axoneme of the new flagellum and a portion of the basal plate of the mature flagellum. Finally, the last electron-dense plate is found between the two axonemes.

The new flagellum is attached to the mature flagellum via a flagella connector structure. The flagella connector is an electron-dense plate structure organized into three distinct layers laterally connecting the distal region of the new flagellum to the mature flagellum.

Number 1 refers to the mature flagellum and number 2 to the new flagellum. Trypanosome duplication is well documented in procyclic and bloodstream forms.

Differentiation from one stage to the other has been well investigated for bloodstream to procyclic conversion [ 39 , 40 , 41 ].

By contrast, trypanosome development in the tsetse PV is still poorly understood [ 28 ]. This is explained by the lack of in vitro culture for parasites from PV and therefore requires the dissection of a large number of flies which is time-consuming and demands experienced staff. In this paper, we revisited the process leading to the asymmetric division and the production of long and short epimastigotes in the tsetse proventriculus Fig.

It was thought that the order of the events was first the differentiation of trypomastigotes into epimastigotes that was previously inferred to follow a series of cellular modifications according to a precise chronological plan: i nucleus migration to the posterior region of the body, followed by ii new flagellum assembly; iii duplication of the kinetoplast; and iv mitosis; and ending with v cytokinesis to produce one long and one short epimastigote cell [ 3 , 10 , 28 ].

Sleeping sickness is curable with medication but is fatal if left untreated. Image: Trypanosoma brucei rhodesiense in a Giemsa-stained blood smear. Credit: DPDx. Contact Us. Skip directly to site content Skip directly to page options Skip directly to A-Z link. Parasites - African Trypanosomiasis also known as Sleeping Sickness.