RESUMO
T cells use their T-cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity foreign peptide major-histocompatibility-complexes (pMHCs) based on the TCR/pMHC off-rate. It is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off-rate remains debated. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity TCR/pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds). This was confirmed by molecular dynamics simulations. Consistent with these findings, we show that the best-characterized catch bond, involving the OT-I TCR, has a low affinity and an exceptionally fast solution off-rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination, and we suggest a role for the adhesion receptors CD2 and LFA-1 in force-shielding the TCR/pMHC interaction.
Assuntos
Receptores de Antígenos de Linfócitos T , Linfócitos T , Receptores de Antígenos de Linfócitos T/metabolismo , Complexo Principal de Histocompatibilidade , Peptídeos , Simulação de Dinâmica Molecular , Ligação ProteicaRESUMO
Binding of the T cell receptor complex to its ligand, the subsequent molecular rearrangement, and the concomitant cell-scale shape changes represent the very first steps of adaptive immune recognition. The first minutes of the interaction of T cells and antigen presenting cells have been extensively scrutinized; yet, gaps remain in our understanding of how the biophysical properties of the environment may impact the sequence of events. In particular, many pioneering experiments were done on immobilized ligands and gave major insights into the process of T cell activation, whereas later experiments have indicated that ligand mobility was of paramount importance, especially to enable the formation of T cell receptor clusters. Systematic experiments to compare and reconcile the two schools are still lacking. Furthermore, recent investigations using compliant substrates have elucidated other intriguing aspects of T cell mechanics. Here we review experiments on interaction of T cells with planar artificial antigen presenting cells to explore the impact of mechanics on adhesion and actin morphodynamics during the spreading process. We enumerate a sequence tracing first contact to final spread state that is consistent with current understanding. Finally, we interpret the presented experimental results in light of a mechanical model that captures all the different morphodynamic states.
Assuntos
Linfócitos T , Linfócitos T/citologia , Humanos , Animais , Fenômenos Biomecânicos , Modelos Biológicos , Adesão Celular , Movimento Celular , Actinas/metabolismoRESUMO
Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.
Assuntos
Acústica , DNA , Proteínas , Análise Espectral , Análise Espectral/métodos , DNA/química , Proteínas/química , Mapas de Interação de Proteínas , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo , Sirolimo/química , Sirolimo/metabolismo , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
The T cell receptor (TCR)-peptide-MHC (pMHC) interaction is the only antigen-specific interaction during T lymphocyte activation. Recent work suggests that formation of catch bonds is characteristic of activating TCR-pMHC interactions. However, whether this binding behavior is an intrinsic feature of the molecular bond, or a consequence of more complex multimolecular or cellular responses, remains unclear. We used a laminar flow chamber to measure, first, 2D TCR-pMHC dissociation kinetics of peptides of various activating potency in a cell-free system in the force range (6 to 15 pN) previously associated with catch-slip transitions and, second, 2D TCR-pMHC association kinetics, for which the method is well suited. We did not observe catch bonds in dissociation, and the off-rate measured in the 6- to 15-pN range correlated well with activation potency, suggesting that formation of catch bonds is not an intrinsic feature of the TCR-pMHC interaction. The association kinetics were better explained by a model with a minimal encounter duration rather than a standard on-rate constant, suggesting that membrane fluidity and dynamics may strongly influence bond formation.
Assuntos
Antígeno HLA-A2/química , Modelos Químicos , Receptores de Antígenos de Linfócitos T/química , Sistema Livre de Células , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Cinética , Ligação Proteica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Mechanosensing by T cells through the T cell receptor (TCR) is at the heart of immune recognition. While the mechanobiology of the TCR at the molecular level is increasingly well documented, its link to cell-scale response is poorly understood. Here we explore T cell spreading response as a function of substrate rigidity and show that remarkably, depending on the surface receptors stimulated, the cellular response may be either biphasic or monotonous. When adhering solely via the TCR complex, T cells respond to environmental stiffness in an unusual fashion, attaining maximal spreading on an optimal substrate stiffness comparable to that of professional antigen-presenting cells. However, in the presence of additional ligands for the integrin LFA-1, this biphasic response is abrogated and the cell spreading increases monotonously with stiffness up to a saturation value. This ligand-specific mechanosensing is effected through an actin-polymerization-dependent mechanism. We construct a mesoscale semianalytical model based on force-dependent bond rupture and show that cell-scale biphasic or monotonous behavior emerges from molecular parameters. As the substrate stiffness is increased, there is a competition between increasing effective stiffness of the bonds, which leads to increased cell spreading and increasing bond breakage, which leads to decreased spreading. We hypothesize that the link between actin and the receptors (TCR or LFA-1), rather than the ligand/receptor linkage, is the site of this mechanosensing.
Assuntos
Mecanotransdução Celular , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Linhagem Celular , Humanos , Imunidade Celular , Cinética , Ligantes , Miosinas/metabolismo , Especificidade por Substrato , Linfócitos T/imunologiaRESUMO
Protein patterning has emerged as a powerful means to interrogate adhering cells. However, the tools to apply a sub-micrometer periodic stimulus and the analysis of the response are still being standardized. We propose a technique combining electron beam lithography and surface functionalization to fabricate nanopatterns compatible with advanced imaging. The repetitive pattern enables a deep-learning algorithm to reveal that T cells organize their membrane and actin network differently depending upon whether the ligands are clustered or homogeneously distributed, an effect invisible to the unassisted human eye even after extensive image analysis. This fabrication and analysis toolbox should be useful, both together and separately, for exploring general correlation between a spatially structured subcellular stimulation and a subtle cellular response.
Assuntos
Inteligência Artificial , Linfócitos T , Humanos , Inteligência , Ligantes , ImpressãoRESUMO
Antibodies are key tools in biomedical research and medicine. Their binding properties are classically measured in solution and characterized by an affinity. However, in physiological conditions, antibodies can bridge an immune effector cell and an antigen-presenting cell, implying that mechanical forces may apply to the bonds. For example, in antibody-dependent cell cytotoxicity-a major mode of action of therapeutic monoclonal antibodies-the Fab domains bind the antigens on the target cell, whereas the Fc domain binds to the activating receptor CD16 (also known as FcgRIII) of an immune effector cell, in a quasi-bidimensional environment (2D). Therefore, there is a strong need to investigate antigen/antibody binding under force (2D) to better understand and predict antibody activity in vivo. We used two anti-CD16 nanobodies targeting two different epitopes and laminar flow chamber assay to measure the association and dissociation of single bonds formed between microsphere-bound CD16 antigens and surface-bound anti-CD16 nanobodies (or single-domain antibodies), simulating 2D encounters. The two nanobodies exhibit similar 2D association kinetics, characterized by a strong dependence on the molecular encounter duration. However, their 2D dissociation kinetics strongly differ as a function of applied force: one exhibits a slip bond behavior in which off rate increases with force, and the other exhibits a catch-bond behavior in which off rate decreases with force. This is the first time, to our knowledge, that catch-bond behavior was reported for antigen-antibody bond. Quantification of natural killer cells spreading on surfaces coated with the nanobodies provides a comparison between 2D and three-dimensional adhesion in a cellular context, supporting the hypothesis of natural killer cell mechanosensitivity. Our results may also have strong implications for the design of efficient bispecific antibodies for therapeutic applications.
Assuntos
Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Fenômenos Mecânicos , Anticorpos de Domínio Único/imunologia , Fenômenos Biomecânicos , Adesão Celular , Linhagem Celular , HumanosRESUMO
Receptors at the membrane of immune cells are the central players of innate and adaptative immunity, providing effective defence mechanisms against pathogens or cancer cells. Their function is intimately linked to their position at and within the membrane which provides accessibility, mobility as well as membrane proximal cytoskeleton anchoring, all of these elements playing important roles in the final function and links to cellular actions. Understanding how immune cells integrate the specific signals received at their membrane to take a decision remains an immense challenge and a very active field of fundamental and applied research. Recent progress in imaging and micromanipulation techniques have led to an unprecedented refinement in the description of molecular structures and supramolecular assemblies at the immune cell membrane, and provided a glimpse into their dynamics and regulation by force. Several key elements have been scrutinized such as the roles of relative sizes of molecules, lateral organisation, motion in the membrane of the receptors, but also physical cues such as forces, mediated by cellular substrates of different rigidities or applied by the cell itself, in conjunction with its partner cell. We review here these recent discoveries associated with a description of the biophysical methods used. While a conclusive picture integrating all of these components is still lacking, mainly due to the implication of diverse and different mechanisms and spatio-temporal scales involved, the amount of quantitative data available opens the way for physical modelling and numerical simulations and new avenues for experimental research.
Assuntos
Membrana Celular/química , Linfócitos/química , Receptores de Antígenos/química , Transdução de Sinais , Animais , Membrana Celular/imunologia , Humanos , Linfócitos/imunologia , Receptores de Antígenos/imunologiaRESUMO
There are very few techniques to reconstruct the shape of a cell at nanometric resolution, and those that exist are almost exclusively based on fluorescence, implying limitations due to staining constraints and artifacts. Reflection interference contrast microscopy (RICM), a label-free technique, permits the measurement of nanometric distances between refractive objects. However, its quantitative application to cells has been largely limited due to the complex interferometric pattern caused by multiple reflections on internal or thin structures like lamellipodia. Here we introduce 3D reflection interference contrast nanoscopy, 3D-RICN, which combines information from multiple illumination wavelengths and aperture angles to characterize the lamellipodial region of an adherent cell in terms of its distance from the surface and its thickness. We validate this new method by comparing data obtained on fixed cells imaged with atomic force microscopy and quantitative phase imaging. We show that as expected, cells adhering to micropatterns exhibit a radial symmetry for the lamellipodial thickness. We demonstrate that the substrate-lamellipod distance may be as high as 100 nm. We also show how the method applies to living cells, opening the way for label-free dynamical study of cell structures with nanometric resolution.
RESUMO
Living cells sense the physical and chemical nature of their micro/nano environment with exquisite sensitivity. In this context, there is a growing need to functionalize soft materials with micro/nanoscale biochemical patterns for applications in mechanobiology. This, however, is still an engineering challenge. Here a new method is proposed, where submicronic protein-patterns are first formed on glass and are then printed on to an elastomer. The degree of transfer is shown to be governed mainly by hydrophobic interactions and to be influenced by grafting an appropriate fluorophore onto the core protein of interest. The transfer mechanism is probed by measuring the forces of adhesion/cohesion using atomic force microscopy. The transfer of functional arrays of dots with size down to about 400 nm, on elastomers with stiffness ranging from 3 kPa to 7 MPa, is demonstrated. Pilot studies on adhesion of T lymphocytes on such soft patterned substrates are reported.
Assuntos
Nanopartículas/química , Soroalbumina Bovina/química , Elastômeros de Silicone/química , Animais , Bovinos , Adesão Celular , Corantes , Dimetilpolisiloxanos/química , Corantes Fluorescentes/química , Vidro/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Nylons/química , Imagem Óptica , Tamanho da Partícula , Poloxâmero/química , Impressão , Propriedades de Superfície , Linfócitos T/fisiologia , XantenosRESUMO
Arrays of protein nanodots with dot-size tuned independently of spacing (e.g., â¼100 to 600 nm diameter for 900 nm spacing) are fabricated. The mechanism of size control is demonstrated, by numerical simulations, to arise from shadow effects during deposition of a sacrificial metal mask. We functionalize the nanodots with antibodies and embed them in a polymer-cushion or in lipid-bilayers or transfer them to soft elastomers. Their ability to influence cell architecture and local membrane organization is demonstrated in T-lymphocytes, using reflection interference contrast and total internal reflection fluorescence microscopy.
RESUMO
Spreading of T cells on antigen presenting cells is a crucial initial step in immune response. Spreading occurs through rapid morphological changes concomitant with the reorganization of surface receptors and of the cytoskeleton. Ligand mobility and frictional coupling of receptors to the cytoskeleton were separately recognized as important factors but a systematic study to explore their biophysical role in spreading was hitherto missing. To explore the impact of ligand mobility, we prepared chemically identical substrates on which molecules of anti-CD3 (capable of binding and activating the T cell receptor complex), were either immobilized or able to diffuse. We quantified the T cell spreading area and cell edge dynamics using quantitative reflection interference contrast microscopy, and imaged the actin distribution. On mobile ligands, as compared to fixed ligands, the cells spread much less, the actin is centrally, rather than peripherally distributed and the edge dynamics is largely altered. Blocking myosin-II or adding molecules of ICAM1 on the substrate largely abrogates these differences. We explain these observations by building a model based on the balance of forces between activation-dependent actin polymerization and actomyosin-generated tension on one hand, and on the frictional coupling of the ligand-receptor complexes with the actin cytoskeleton, the membrane and the substrate, on the other hand. Introducing the measured edge velocities in the model, we estimate the coefficient of frictional coupling between T Cell receptors or LFA-1 and the actin cytoskeleton. Our results provide for the first time, to our knowledge, a quantitative framework bridging T cell-specific biology with concepts developed for integrin-based mechanisms of spreading.
Assuntos
Movimento Celular , Forma Celular , Fricção , Linfócitos T/citologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat , Ligantes , Miosina Tipo II/metabolismo , Pseudópodes/metabolismo , Linfócitos T/metabolismoRESUMO
We present a simple cost-effective benchtop protocol to functionalize glass and polydimethylsiloxane (PDMS) with nanometric protein patches for cell adhesion studies. Evaporation masks, covering macroscopic areas on glass, were made using improved strategies for self-assembly of colloidal microbeads which then served as templates for creating the protein patch arrays via the intermediate steps of organo-aminosilane deposition and polyethylene-glycol grafting. The diameter of the patches could be varied down to about 80 nm. The glass substrates were used for advanced optical imaging of T-lymphocytes to explore adhesion by reflection interference contrast microscopy and the possible colocalization of T-cell receptor microclusters and the activating protein patches by total internal reflection fluorescence microscopy. The selectively functionalized glass could also serve as template for transferring the protein nanopatches to the surface of a soft elastomer. We demonstrated successful reverse contact printing onto the surface of thin layers of PDMS with stiffness ranging from 30 KPa to 3 MPa.
Assuntos
Adesão Celular/fisiologia , Dimetilpolisiloxanos/química , Vidro/química , Nanopartículas/química , Soroalbumina Bovina/química , Soroalbumina Bovina/farmacocinética , Linfócitos T/fisiologia , Adsorção , Separação Celular/métodos , Células Cultivadas , Humanos , Células Jurkat , Teste de Materiais , Nanopartículas/ultraestrutura , Tamanho da Partícula , Propriedades de Superfície , Linfócitos T/citologiaRESUMO
In vitro display technologies such as yeast display have been instrumental in developing the selection of new antibodies, antibody fragments or nanobodies that bind to a specific target, with affinity towards the target being the main factor that influences selection outcome. However, the roles of mechanical forces are being increasingly recognized as a crucial factor in the regulation and activation of effector cell function. It would thus be of interest to isolate binders behaving optimally under the influence of mechanical forces. We developed a microfluidic assay allowing the selection of yeast displaying nanobodies through antigen-specific immobilization on a surface under controlled hydrodynamic flow. This approach enabled enrichment of model yeast mixtures using tunable antigen density and applied force. This new force-based selection method opens the possibility of selecting binders by relying on both their affinity and force resistance, with implications for the design of more efficient immunotherapeutics.
Assuntos
Antígenos , Saccharomyces cerevisiae , Anticorpos de Domínio Único , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/imunologia , Saccharomyces cerevisiae/metabolismo , Antígenos/imunologia , Antígenos/metabolismo , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
The role of force application in immune cell recognition is now well established, the force being transmitted between the actin cytoskeleton to the anchoring ligands through receptors such as integrins. In this chain, the mechanics of the cytoskeleton to receptor link, though clearly crucial, remains poorly understood. To probe this link, we combine mechanical extraction of membrane tubes from T cells using optical tweezers, and fitting of the resulting force curves with a viscoelastic model taking into account the cell and relevant molecules. We solicit this link using four different antibodies against various membrane bound receptors: antiCD3 to target the T Cell Receptor (TCR) complex, antiCD45 for the long sugar CD45, and two clones of antiCD11 targeting open or closed conformation of LFA1 integrins. Upon disruption of the cytoskeleton, the stiffness of the link changes for two of the receptors, exposing the existence of a receptor to cytoskeleton link-namely TCR-complex and open LFA1, and does not change for the other two where a weaker link was expected. Our integrated approach allows us to probe, for the first time, the mechanics of the intracellular receptor-cytoskeleton link in immune cells.
Assuntos
Citoesqueleto , Nanotubos , Microtúbulos , Receptores Imunológicos , Integrinas , Receptores de Antígenos de Linfócitos TRESUMO
The binding properties of biomolecules play a crucial role in many biological phenomena, especially cell adhesion. Whereas the attachment kinetics of soluble proteins is considered well known, complex behavior arises when protein molecules are bound to the cell membrane. We probe the hidden kinetics of ligand-receptor bond formation using single-molecule flow chamber assays and Brownian dynamics simulations. We show that, consistent with our recently proposed hypothesis, association requires a minimum duration of contact between the reactive species. In our experiments, ICAM-1 anchored on a flat substrate binds to anti-ICAM-1 coated onto flowing microbeads. The interaction potential between bead and substrate is measured by microinterferometry and is used as an ingredient to simulate bead movement. Our simulation calculates the duration of ligand-receptor contacts imposed by the bead movement. We quantitatively predict the reduction of adhesion probability measured for shorter tether length of the ligand or if a repulsive hyaluronan layer is added onto the surface. To account for our results, we propose that bond formation may occur in our system by crossing of a diffusive plateau in the energy landscape, on the timescale of 5 ms and an energy barrier of 5 k(B)T, before reaching the first detectable bound state. Our results show how to relate cell-scale behavior to the combined information of molecular reactivity and biomolecule submicron-scale environment.
Assuntos
Molécula 1 de Adesão Intercelular/imunologia , Adsorção , Animais , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Fenômenos Biomecânicos , Materiais Biomiméticos/química , Adesão Celular , Glicocálix/química , Humanos , Molécula 1 de Adesão Intercelular/química , Ligantes , Microesferas , Polímeros/química , Propriedades de Superfície , Fatores de TempoRESUMO
The scanning of surrounding tissues by T lymphocytes to detect cognate antigens requires high speed, sensitivity and specificity. T-cell receptor (TCR) co-receptors such as CD8 increase detection performance, but the exact mechanism remains incompletely understood. Here, we used a laminar flow chamber to measure at the single molecule level the kinetics of bond formation and rupture between TCR- transfected CD8+ and CD8- Jurkat cells and surfaces coated with five peptide-exposing major histocompatibility antigens (pMHCs) of varying activating power. We also used interference reflection microscopy to image the spreading of these cells dropped on pMHC-exposing surfaces. CD8 did not influence the TCR-pMHC interaction during the first few seconds following cell surface encounter, but it promoted the subsequent spreading responses, suggesting that CD8 was involved in early activation rather than binding. Further, the rate and extent of spreading, but not the lag between contact and spreading initiation, depended on the pMHC. Elucidating T-lymphocyte detection strategy may help unravel underlying signaling networks.
Assuntos
Antígenos CD8/metabolismo , Microscopia de Interferência/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , HumanosRESUMO
We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping to control at the same time the position, shape, spreading, mechanics and activation state of T cells. Once adhered on patterns, we examined the capacities of T cells to be activated with soluble anti CD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 antibody decorated surface was not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. Aside, we analyzed the T cell mechanics, when spread on pattern or not, using Atomic Force Microscopy indentation. By expressing MEGF10 as a non immune adhesion receptor in T cells we measured the very same spreading area on PLL substrates and Young modulus than non modified cells, immobilized on anti CD45 antibodies, while retaining similar activation capabilities using soluble anti CD3 antibodies or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenized populations in standardized T cell activation assays.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Biomarcadores , Cálcio/metabolismo , Adesão Celular/imunologia , Linhagem Celular Tumoral , Forma Celular , Expressão Ectópica do Gene , Humanos , Antígenos Comuns de Leucócito/metabolismo , Imagem MolecularRESUMO
We examine experimental and theoretical aspects of nonspecific adhesion of giant vesicles on modified surfaces as model systems for cell spreading. Using dual-wave interference microscopy and new analysis, membrane undulations as well as large scale vesicle shape are monitored. Measurements and modelling show that the nucleation of adhesion depends critically on the interfacial polymer and membrane tension. Patch growth is governed by local membrane geometry, adhesion energy, and local viscosity. Finally, spreading stops when tension induced by adhesion unfolds excess membrane area.
Assuntos
Polímeros/metabolismo , Lipossomas Unilamelares/metabolismo , Adesividade , Adesão Celular , Modelos Biológicos , Tensão SuperficialRESUMO
Oocysts are the environmentally resistant stage of the protozoan parasite Toxoplasma gondii. They are responsible for foodborne infections in humans and animals worldwide. Infectious oocysts contain sporozoites that have to exit the sporocyst and oocyst walls to initiate replication of the parasite within the host tissues. Given their robustness and resistance to chemical degradation, it is still unclear how the oocyst and sporocyst walls release the sporozoites. This process called excystation is thought to occur in the small intestine as a result of the combined action of digestive agents, yet to be identified. By using an oocyst-macrophage co-culture platform, we previously demonstrated in vitro that the excystation of sporozoites and their differentiation into replicative tachyzoites could occur in absence of digestive factors, following phagocytosis by macrophages. Here, we further characterize the dynamics of the oocyst phagocytosis at the single-cell level by using optical tweezers and micropipette aspiration techniques. Our results show that the oocyst internalization kinetics can vary among a given population of macrophages, but similar processes and dynamics could be observed. Most of the cells manipulate oocysts for ~15 min before internalizing them in typically 30 min. This process mainly involves the actin cytoskeleton of the macrophages. Liberated sporozoites within macrophages then differentiate into tachyzoites within 4-6 h following oocyst-macrophage contact. Tachyzoites appear to develop better in macrophages challenged with free sporocysts or sporozoites than with whole oocysts, suggesting that opening of the oocyst wall is one of the most limiting steps for sporozoite excystation completion.