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This paper presents a segmentation and tracking method for quantitative analysis of cell dynamics from in vitro videomicroscopy data. The method is based on parametric active contours and includes several adaptations that address important difficulties of cellular imaging, particularly the presence of low-contrast boundary deformations known as pseudopods, and the occurence of multiple contacts between cells. First, we use an edge map based on the average intensity dispersion that takes advantage of relative background homogeneity to facilitate the detection of both pseudopods and interfaces between adjacent cells. Second, we introduce a repulsive interaction between contours that allows correct segmentation of objects in contact and overcomes the shortcomings of previously reported techniques to enforce contour separation. Our tracking technique was validated on a realistic data set by comparison with a manually defined ground-truth and was successfully applied to study the motility of amoebae in a biological research project.

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demonstrated by a co-sedimentation assay. A variant of EhCaBP1 did not bind F-actin showing the specificity of the interaction between EhCaBP1 and actin. There is no significant change in the kinetics of in vitro polymerization of actin in presence of EhCaBP1, indicating that EhCaBP1 does not affect filament treadmilling. In addition, using atomic force microscopy; it was found that filaments of Factin, polymerized in presence of EhCaBP1, were thinner. These results indicate that EhCaBP1 may be involved in dynamic membrane restructuring at the time of cell pseudopod formation, phagocytosis and endocytosis in a process mediated by direct binding of EhCaBP1 to actin, affecting the bundling of actin filaments.

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.

SummaryPhagocytosis of human cells is a crucial activity for the virulence of the human parasite Entamoeba histolytica . This protozoan invades and destroys the intestine by killing and phagocytosing epithelial cells, erythrocytes and cells from the immune system. In this study, we used magnetic beads covered with proteins from human serum as a model system to study the early events involved in phagocytosis by E. histolytica . We validated the system showing that the beads uptake triggered the activation of the actinmyosin cytoskeleton and involved a PI3-kinase as previously described for erythrophagocytosis. We purified early phagosomes from wild-type (WT) amoeba and from parasites that overproduced myosin IB (MyoIB + + + + ), the unique unconventional myosin of E. histolytica . The MyoIB + + + + cells exhibit a slower and more synchronized uptake process than the WT strain. Proteomic analysis by liquid chromatography and tandem mass spectroscopy (LC-MS/MS) of the WT and MyoIB + + + + phagosomes allowed us to identify, for the first time, molecular actors involved in the early step of the uptake process. These include proteins involved in cytoskeleton activity, signalling, endocytosis, lytic activity and cell surface proteins. Interestingly, the proteins that we found specifically recruited on the phagosomes from the MyoIB + + + + strain were previously described in other eukarytotic cells, as involved in the regulation of cortical F-actin dynamics, such as a a a a -actinin and formins. This proteomics approach allows a step further towards the understanding of the molecular mechanisms involved in phagocytosis in E. histolytica that revealed some interesting differences compared with phagocytosis in macrophages or Dictyostelium discoideum , and allowed to identify putative candidates for proteins linked to myosin IB activity during the phagocytic process

Variational deformable models have proven over the past decades a high efficiency for segmentation and tracking in 2-D sequences. Yet, their application to 3-D time-lapse images has been hampered by discretization issues, heavy computational loads and lack of proper user visualization and interaction, limiting their use for routine analysis of large data-sets. We propose here to address these limitations by reformulating the problem entirely in the discrete domain using 3-D active meshes, which express a surface as a discrete triangular mesh, and minimize the energy functional accordingly. By performing computations in the discrete domain, computational costs are drastically reduced, whilst the mesh formalism allows to benefit from real-time 3-D rendering and other GPU-based optimizations. Performance evaluations on both simulated and real biological data sets show that this novel framework outperforms current state-of-the-art methods, constituting a light and fast alternative to traditional variational models for segmentation and tracking applications.

SummaryWe have demonstrated that the two-and three-dimensional motility of the human pathogenic parasite Entamoeba histolytica (Eh) depends on sustained instability of the intracellular hydrostatic pressure. This instability drives the cyclic generation and healing of membrane blebs, with typical protrusion velocities of 10-20 m/second over a few hundred milliseconds and healing times of 10 seconds. The use of a novel micro-electroporation method to control the intracellular pressure enabled us to develop a qualitative model with three parameters: the rate of the myosin-driven internal pressure increase; the critical disjunction stress of membrane-cytoskeleton bonds; and the turnover time of the F-actin cortex. Although blebs occur randomly in space and irregularly time, they can be forced to occur with a defined periodicity in confined geometries, thus confirming our model. Given the highly efficient bleb-based motility of Eh in vitro and in vivo, Eh cells represent a unique model for studying the physical and biological aspects of amoeboid versus mesenchymal motility in two-and three-dimensional environments.