A Quantitative 3D Motility Analysis of Trypanosoma brucei by Use of Digital In-line Holographic Microscopy

We present a quantitative 3D analysis of the motility of the blood parasite Trypanosoma brucei. Digital in-line holographic microscopy has been used to track single cells with high temporal and spatial accuracy to obtain quantitative data on their behavior. Comparing bloodstream form and insect form trypanosomes as well as mutant and wildtype cells under varying external conditions we were able to derive a general two-state-run-and-tumble-model for trypanosome motility. Differences in the motility of distinct strains indicate that adaption of the trypanosomes to their natural environments involves a change in their mode of swimming

Cell biological aspects of motility of Trypanosoma brucei in consideration of the interaction with the microenvironment

Trypanosomen sind Protozoen, die Krankheiten bei Mensch und Tier verursachen, die unbehandelt infaust verlaufen. Die Zellen sind hoch motil, angetrieben von einem einzelständigen Flagellum, welches entlang des Zellkörpers angeheftet ist. Selbst in Zellkultur hören Trypanosomen niemals auf sich zu bewegen und eine Ablation funktioneller Bestandteile des Flagellarapparates ist letal für Blutstromformen. Es wurde gezeigt, dass Motilität notwendig ist für die Zellteilung, Organellenpositionierung und Infektiosität. Dies macht Trypanosomen zu besonders geeigneten Modellorganismen für die Untersuchung der Motilität. Dennoch ist erstaunlich wenig über die Motilität bei Trypanosomen bekannt. Dies gilt auch noch genereller für die Protozoen. Unlängst ist dieses Gebiet allerdings in den Fokus vieler Arbeiten gerückt, was bereits erstaunliche, neue Erkenntnisse hervorgebracht hat. Doch Vieles ist noch nicht abschliessend geklärt, so z.B. wie der Flagellarschlag genau reguliert wird, oder wie sich der Schlag des Flagellums entlang des Zellkörpers ausbreitet. Die vorliegende Arbeit befasst sich besonders mit den Einflüssen, die die Mikroumgebung auf die Motilität von Blutstromform-Trypanosomen ausübt. In ihrem natürlichen Lebensraum finden sich Trypanosomen in einer hoch komplexen Umgebung wieder. Dies gilt sowohl für den Blutkreislauf, als auch für den Gewebezwischenraum in ihrem Säugerwirt. Die hohe Konzentration von Zellen, Gewebeverbänden und extrazellulären Netzwerken könnte man als Ansammlung von Hindernissen für die Fortbewegung auffassen. Diese Arbeit zeigt dagegen, dass der Mechanismus der Bewegung eine Adaptation an genau diese Umweltbedingungen darstellt, so z.B. an die Viskosität von Blut. Es wird auch ein Bewegungsmodell vorgestellt, das erläutert, worin diese Adaption besteht. Dies erklärt auch, warum die Mehrheit der Zellen einer Trypanosomenkultur eine ungerichtete Taumel-Bewegung aufweist in nieder-viskosem Medium, das keine solchen “Hindernisse” enthält. Die Zugabe von Methylcellulose in einer Konzentration von ca. 0,5% (w/v) erwies sich als geeigneter Ersatz von Blut, um optimale Bedingungen für gerichtetes Schwimmen von Blutstromform Trypanosomen zu erreichen. Zusätzlich wurden in dieser Arbeit unterschiedliche Arten von Hindernissen, wie Mikroperlen (Beads) oder molekulare Netzwerke, sowie artifizielle, geordnete Mikrostrukturen verwendet, um die Interaktion mit einer festen Matrix zu untersuchen. In deren Anwesenheit war sowohl die Schwimmgeschwindigkeit, als auch der Anteil an persistent schwimmenden Trypanosomen erhöht. Zellen, die frei schwimmend in Flüssigkeiten vorkommen (wie Euglena oder Chlamydomonas), werden effizient durch einen planaren Schlag des Flagellums angetrieben. Trypanosomen hingegen mussten sich evolutionär an eine komplexe Umgebung anpassen, die mit einer zu raumgreifenden Welle interferieren würde. Der dreidimensionale Flagellarschlag des, an die Zelloberfläche angehefteten, Flagellums erlaubt den Trypanosomen eine effiziente Fortbewegung durch die Interaktion mit Objekten in jedweder Richtung gleichermassen. Trypanosomen erreichen dies durch eine hydrodynamisch verursachte Rotation ihres Zellkörpers entlang ihrer Längsachse, entgegen dem Uhrzeigersinn. Der Einfluss der Mikroumgebung wurde in früheren Untersuchungen bisher vernachlässigt, ist zum Verständnis der Motilität von T. brucei jedoch unerlässlich. Ein weiterer, bisher nicht untersuchter Aspekt der Beeinflussung der Motilität durch die Umwelt sind hydrodynamische Strömungseffekte, denen Trypanosomen im kardiovaskulären System ausgesetzt sind. Diese wurden in dieser Arbeit mittels Mikrofluidik untersucht. Um unser Verständnis der Motilität von Trypanosomen von 2D, wie üblich in der Motilitätsanalyse mittels Lebend-Zell-Mikroskopie, auf drei Dimensionen auszudehnen, wurde als bildgebendes Verfahren auch die Holographie eingesetzt. Mikrofluidik und Holographie sind beides aufkommende Techniken mit großem Anwendungspotential in der Biologie, die zuvor noch nie für die Motilitätsanalyse von Trypanosomen eingesetzt worden waren. Dies erforderte daher interdisziplinäre Kooperationen. Zusätzlich wurde in dieser Arbeit auch ein vollständig automatisiertes und Software-gesteuertes Fluoreszenzmikroskopiesystem entwickelt, das in der Lage ist, einzelne Zellen durch entsprechende Steuerung des Mikroskoptisches autonom zu verfolgen und somit eine Bewegungsanalyse in Echtzeit ermöglicht, ohne weitere Benutzerinteraktion. Letztendlich konnte dadurch auch die Bewegung der schlagenden Flagelle und des gesamten Zellkörpers mit hoher zeitlicher und räumlicher Auflösung mittels Hochgeschwindigkeits-Fluoreszenzmikroskopie aufgeklärt werden.Trypanosomes are protozoa causing fatal diseases in livestock and man. The cells show vivid motility, driven by a single flagellum that runs along the cell body, attached to the cell surface. Even in cell culture, trypanosomes never stop moving and ablation of functional components of the flagellum is lethal for bloodstream-forms. Motility has been shown to be essential for cell division, organelle positioning and infectivity. This renders trypanosomes valuable model organisms for studying motility. But, surprisingly little is known about motility in trypanosomes, as well as in protozoa, in general. Recently, motility of trypanosomes therefore has gotten into the spotlight of interest which brought some new insights, but many essential points are still a matter of debate, for example how the flagellar beat is regulated or how it is propagated along the cell body. In this work, the effects of the micro-environment of blood-stream form trypanosomes on motility were investigated. In their natural habitat, trypanosomes find themselves in a crowded environment. This is not only the case in the blood circulatory system, but also in extra-tissue space. The high concentration of cells and extra-cellular networks might be regarded as a kind of obstacle to cellular motion. This work shows that the mode of motility of bloodstream form trypanosomes instead is adapted to the viscosity of blood. Also a mechanistic model is presented which elucidates how this adaptation works. This also explains why most trypanosomes are tumbling in low-viscous cell culture medium, lacking other cellular components. Addition of Methylcellulose at a concentration of about 0.5% (w/v) was found to be a potent substitute for blood, providing optimal conditions for trypanosome motility. Also different types of obstacles like beads and molecular networks, as well as arranged pillar microstructures were used as a tool to mimic interaction with a solid matrix. In presence of these, the swimming speed as well as the percentage of persistent swimming cells was increased. Cells inhabiting an open-ranged environment (like Euglena or Chlamydomonas) are efficiently propelled by a planar flagellar wave. Trypanosomes in contrast, had to evolutionary adapt to a crowded environment, which would infer with any extensive planar wave. The three-dimensional flagellar beat of the attached flagellum allows trypanosomes to harness any rigid matrix for effective propulsion, in all directions equally. Trypanosomes achieve this by a rotational counter-clockwise motion of their whole cell body. Another environmental aspect for trypanosome motility that had not been studied before is the influence of hydrodynamic flow, which trypanosomes are subjected to, when swimming in the blood circulatory system. For studying this, in this work, the motilty of trypanosomes was analyzed in microfluidic devices. To extend our understanding of trypanosomal motility from 2D, like in standard microscopy based live-cell imaging analysis, to 3D, a imaging technique known as holography was used, in addition. Microfluidics as well as Holography both are emerging, high-potential techniques in biology, which had not been used for the motility analysis of trypanosomes before and establishing this therefore only got possible due to interdisciplinary collaborations. In addition, a custom fully automated, software-controlled, fluorescence microscopic system was developed in this work, which is able to track and follow single cells for motility analysis in real-time without the need for user input. The motion of the flagellar beat and the cell itself was investigated at high spatio-temporal resolution using highspeed fluorescence microscopy

Optical trapping reveals propulsion forces, power generation and motility efficiency of the unicellular parasites Trypanosoma brucei brucei

Unicellular parasites have developed sophisticated swimming mechanisms to survive in a wide range of environments. Cell motility of African trypanosomes, parasites responsible for fatal illness in humans and animals, is crucial both in the insect vector and the mammalian host. Using millisecond-scale imaging in a microfluidics platform along with a custom made optical trap, we are able to confine single cells to study trypanosome motility. From the trapping characteristics of the cells, we determine the propulsion force generated by cells with a single flagellum as well as of dividing trypanosomes with two fully developed flagella. Estimates of the dissipative energy and the power generation of single cells obtained from the motility patterns of the trypanosomes within the optical trap indicate that specific motility characteristics, in addition to locomotion, may be required for antibody clearance. Introducing a steerable second optical trap we could further measure the force, which is generated at the flagellar tip. Differences in the cellular structure of the trypanosomes are correlated with the trapping and motility characteristics and in consequence with their propulsion force, dissipative energy and power generation

Impact of Microscopic Motility on the Swimming Behavior of Parasites : Straighter Trypanosomes are More Directional

Microorganisms, particularly parasites, have developed sophisticated swimming mechanisms to cope with a varied range of environments. African Trypanosomes, causative agents of fatal illness in humans and animals, use an insect vector (the Tsetse fly) to infect mammals, involving many developmental changes in which cell motility is of prime importance. Our studies reveal that differences in cell body shape are correlated with a diverse range of cell behaviors contributing to the directional motion of the cell. Straighter cells swim more directionally while cells that exhibit little net displacement appear to be more bent. Initiation of cell division, beginning with the emergence of a second flagellum at the base, correlates to directional persistence. Cell trajectory and rapid body fluctuation correlation analysis uncovers two characteristic relaxation times: a short relaxation time due to strong body distortions in the range of 20 to 80 ms and a longer time associated with the persistence in average swimming direction in the order of 15 seconds. Different motility modes, possibly resulting from varying body stiffness, could be of consequence for host invasion during distinct infective stages

African Trypanosomes as model system for functional analyses of microbial motility

The locomotion of microorganisms in a microscopic world, where cells move through a fluid environment without using inertial forces, is a fascinating phenomenon in life science. Nature offers clever and inspiring strategies for self-propelling in an environment of no inertia. The flagellate African trypanosome, which causes African sleeping sickness, moves with help of a flagellum, which is firmly attached to its cell body. The beating flagellum leads to a strong distortion of the cell body and therefore to a swimming agitation of trypanosomes. We have found that trypanosomes use a hydrodynamic mechanism to defend against host’s immune attacks. Owing to continuous and directional swimming, host-derived antibodies attached to surface glycoproteins of the cell are dragged to the posterior cell pole, where they are rapidly internalized and destroyed. In the following we present new methodology and techniques to quantify the movements of proteins and the motility of cells. Moreover trypanosome motility schemes and their influence on cellular lifestyle and survival strategies are characterized

Flow loading induces oscillatory trajectories in a bloodstream parasite

The dynamics of isolated microswimmers are studied in bounded flow using the African trypanosome, a unicellular parasite, as the model organism. With the help of a microfluidics platform, cells are subjected to flow and found to follow an oscillatory path that is well fit by a sine wave. The frequency and amplitudes of the oscillatory trajectories are dependent on the flow velocity and cell orientation. When traveling in such a manner, trypanosomes orient upstream while downstream-facing cells tumble within the same streamline. A comparison with immotile trypanosomes demonstrates that self-propulsion is essential to the trajectories of trypanosomes even at flow velocities up to ?40 times higher than their own swimming speed. These studies reveal important swimming dynamics that may be generally pertinent to the transport of microswimmers in flow and may be relevant to microbial pathogenesis

Comparison of switching behavior for BSF and PCF trypanosomes.

<p>A) Mean duration of segments for the swimmer and tumbler state B) Distribution of trypanosome swimming vectors contributing to either the swimming or the tumbling state.</p

Schematic representation of the digital in-line holographic microscopy setup.

<p>A) Schematic drawing of the optical setup used in this study. B) Schematic drawing of the beam path between aperture and detector.</p

Example for switching trypanosome trajectories from the BSF 37°C dataset.

<p>A) shows exemplary trajectories color coded with blue colors indicating fast segments and red colors indicating slower segments. B) Shows the whole class’ velocity distribution with a characteristic dip at 5 µm/s.</p