Markus Engstler

Hydrodynamic Flow-Mediated Protein Sorting on the Cell Surface of Trypanosomes

The unicellular parasite Trypanosoma brucei rapidly removes host-derived immunoglobulin (Ig) from its cell surface, which is dominated by a single type of glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG). We have determined the mechanism of antibody clearance and found that Ig-VSG immune complexes are passively sorted to the posterior cell pole, where they are endocytosed. The backward movement of immune complexes requires forward cellular motility but is independent of endocytosis and of actin function. We suggest that the hydrodynamic flow acting on swimming trypanosomes causes directional movement of Ig-VSG immune complexes in the plane of the plasma membrane, that is, immunoglobulins attached to VSG function as molecular sails. Protein sorting by hydrodynamic forces helps to protect trypanosomes against complement-mediated immune destruction in culture and possibly in infected mammals but likewise may be of functional significance at the surface of other cell types such as epithelial cells lining blood vessels.

Kinetics of endocytosis and recycling of the GPI-anchored variant surface glycoprotein in Trypanosoma brucei

The dense coat of glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) covering parasitic African trypanosomes is essential for survival in mammalian hosts. VSG is internalised and recycled exclusively via a specialised part of the plasma membrane, the flagellar pocket. Direct measurement of the kinetics of VSG endocytosis and recycling shows that the VSG cell-surface pool is turned over within 12 minutes. Correspondingly, the turnover of the intracellular pool (9±4% of total VSG) requires only 1 minute, and this is an exceptionally high rate considering that endocytosis and exocytosis are limited to only 5% of the cell surface area. Kinetic 3D co-localisation analysis using biotinylated VSG and a panel of compartmental markers provides consistent evidence for the itinerary of VSG through the cell: VSG is endocytosed in large clathrin-coated vesicles, which bud from the flagellar pocket membrane at a rate of 6-7 vesicles per second, and is then delivered to RAB5-positive early endosomes. From there, VSG is recycled to RAB11-positive recycling endosomes at two stages, either directly or via RAB7-positive, late endosomes. Small clathrin-coated vesicles carrying fluid-phase cargo and being depleted of VSG bud from early and recycling endosomes. These vesicles are postulated to deliver their content to late endosomes and/or the lysosome. The recycling endosomes give rise to RAB11-positive exocytic carriers that fuse with the flagellar pocket and thereby return VSG to the cell surface. VSG recycling provides an interesting model for studies on the cellular trafficking and sorting of GPI-anchored proteins.

Endocytosis, membrane recycling and sorting of GPI‐anchored proteins: Trypanosoma brucei as a model system

In the flagellated protozoon Trypanosoma brucei, endo‐ and exocytosis are restricted to a small area of the plasma membrane, the flagellar pocket. All endosomal compartments and the single Golgi complex are located within the posterior part of the cell between the flagellar pocket and the nucleus. The use of reverse genetic tools, including RNA interference, in combination with quantitative 3D‐fluorescence and electron microscopic techniques has provided an insight into endosomal membrane traffic, which occurs at a very high rate and appears to exhibit a lower level of complexity than in mammalian cells. The flagellate is an excellent model system for studies on endocytosis, sorting and recycling of glycosylphosphatidylinositol‐anchored glycoproteins, because 107 molecules of the variant surface glycoprotein form a dense coat at the cells surface. Because the endocytic rate varies widely at different stages in the parasites life cycle, trypanosomes may be used for investigating developmental aspects of their endocytic system.

Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest

Mitochondrial fission is mediated by dynamin-like proteins (DLPs). Trypanosoma brucei contains a single DLP, which is the only member of the dynamin superfamily. We have previously shown that expression of the human proapoptotic Bax in T. brucei induces extensive mitochondrial fragmentation. Here we report that Baxinduced mitochondrial fission is abolished in cell lines lacking functional DLP suggesting that the protein is also required for mitochondrial division during the cell cycle. Furthermore, DLP-ablated cells are deficient for endocytosis and as a consequence accumulate enlarged flagellar pockets. Thus, besides its expected role in mitochondrial fission the trypanosomal DLP is required for endocytosis, a function thought to be restricted to classical dynamins. In agreement with its dual function, the DLP localizes to both the mitochondrion and the flagellar pocket, the site where endocytosis occurs. Unexpectedly, ablation of DLP also causes an arrest of cytokinesis. The fact that no multinucleation is observed in the arrested cells argues for a precise cell-cycle block. Furthermore, analysis of a clathrin-knockdown cell line suggests that the cytokinesis arrest is not due to the endocytosis defect. Thus, our results support a working model in which mitochondrial fission triggers a checkpoint for cytokinesis.

Accumulation of a GPI-Anchored Protein at the Cell Surface Requires Sorting at Multiple Intracellular Levels

Proteins modified by glycosylphosphatidylinositol membrane anchors have become popular for investigating the role of membrane lipid microdomains in cellular sorting processes. To this end, trypanosomatids offer the advantage that they express these molecules in high abundance. The parasitic protozoan Trypanosoma brucei is covered by a dense and nearly homogeneous coat composed of a glycosylphosphatidylinositol‐anchored protein, the variant surface glycoprotein, which is essential for survival of the parasite in the mammalian blood. Therefore, T. brucei must possess mechanisms to selectively and efficiently deliver variant surface glycoprotein to the cell surface. In this study, we have quantified the steady‐state distribution of variant surface glycoprotein by differential biotinylation, by fluorescence microscopy and by immunoelectron microscopy on high‐pressure frozen and freeze‐substituted samples. These three techniques provide very similar estimates of the fraction of variant surface glycoprotein located on the cell surface, on average 89.4%. The intracellular variant surface glycoprotein (10.6%) is predominantly located in the endosomal compartment (75%), while 25% are associated with the endoplasmic reticulum, Golgi apparatus and lysosomes. The density of variant surface glycoprotein in the plasma membrane including the membrane of the flagellar pocket, the only site for endo‐ and exocytosis in this organism, is 48–52 times higher than the density in endoplasmic reticulum membranes. The relative densities of the Golgi complex and of the endosomes are 2.7 and 10.8, respectively, compared to the endoplasmic reticulum. This data set provides the basis for an analysis of the dynamics of sorting. Depending on the intracellular itinerary of newly formed variant surface glycoprotein, the high surface density is achieved in two (endoplasmic reticulum → Golgi complex → cell surface) or three enrichment steps (endoplasmic reticulum → Golgi complex → endosomes → cell surface), suggesting sorting between several membrane compartments.

Trypanosome Motion Represents an Adaptation to the Crowded Environment of the Vertebrate Bloodstream

Blood is a remarkable habitat: it is highly viscous, contains a dense packaging of cells and perpetually flows at velocities varying over three orders of magnitude. Only few pathogens endure the harsh physical conditions within the vertebrate bloodstream and prosper despite being constantly attacked by host antibodies. African trypanosomes are strictly extracellular blood parasites, which evade the immune response through a system of antigenic variation and incessant motility. How the flagellates actually swim in blood remains to be elucidated. Here, we show that the mode and dynamics of trypanosome locomotion are a trait of life within a crowded environment. Using high-speed fluorescence microscopy and ordered micro-pillar arrays we show that the parasites mode of motility is adapted to the density of cells in blood. Trypanosomes are pulled forward by the planar beat of the single flagellum. Hydrodynamic flow across the asymmetrically shaped cell body translates into its rotational movement. Importantly, the presence of particles with the shape, size and spacing of blood cells is required and sufficient for trypanosomes to reach maximum forward velocity. If the density of obstacles, however, is further increased to resemble collagen networks or tissue spaces, the parasites reverse their flagellar beat and consequently swim backwards, in this way avoiding getting trapped. In the absence of obstacles, this flagellar beat reversal occurs randomly resulting in irregular waveforms and apparent cell tumbling. Thus, the swimming behavior of trypanosomes is a surprising example of micro-adaptation to life at low Reynolds numbers. For a precise physical interpretation, we compare our high-resolution microscopic data to results from a simulation technique that combines the method of multi-particle collision dynamics with a triangulated surface model. The simulation produces a rotating cell body and a helical swimming path, providing a functioning simulation method for a microorganism with a complex swimming strategy.

ALBA proteins are stage regulated during trypanosome development in the tsetse fly and participate in differentiation

The protozoan parasite Trypanosoma brucei is responsible for sleeping sickness and alternates between mammal and tsetse fly hosts. Two proteins of the ALBA family associate to mRNA in cytoplasmic granules during starvation stress, are stage regulated, and contribute to trypanosome development in the tsetse fly.

The VSG C-terminal domain is inaccessible to antibodies on live trypanosomes.

Graphical abstract The VSG coat of Trypanosoma brucei prevents access of antibodies to the VSG C-terminal domain. Research highlights ▶ Antisera raised against recombinant VSG C-terminal domains. ▶ Anti-VSG C-terminal domain sera recognise fixed but not live cells. ▶ Support for model where diffusion barrier is at the base of the VSG N-terminal domain.

New Approaches to the Microscopic Imaging of Trypanosoma brucei

Protozoan parasites are fearsome pathogens responsible for a substantial proportion of human mortality, morbidity, and economic hardship. The principal disease agents are members of the orders Apicomplexa (Plasmodium, Toxoplasma, Eimeria) and Kinetoplastida (Trypanosomes, Leishmania). The majority of humans are at risk from infection from one or more of these organisms, with profound effects on the economy, social structure and quality of life in endemic areas; Plasmodium itself accounts for over one million deaths per annum, and an estimated 4 x 10(7) disability-adjusted life years (DALYs), whereas the Kinetoplastida are responsible for over 100,000 deaths per annum and 4 x 10(6) DALYs. Current control strategies are failing due to drug resistance and inadequate implementation of existing public health strategies. Trypanosoma brucei, the African Trypanosome, has emerged as a favored model system for the study of basic cell biology in Kinetoplastida, because of several recent technical advances (transfection, inducible expression systems, and RNA interference), and these advantages, together with genome sequencing efforts are widely anticipated to provide new strategies of therapeutic intervention. Here we describe a suite of methods that have been developed for the microscopic analysis of T. brucei at the light and ultrastructural levels, an essential component of analysis of gene function and hence identification of therapeutic targets.

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.