Plasmodium falciparum maturation across the intra-erythrocytic cycle shifts the soft glassy viscoelastic properties of red blood cells from a liquid-like towards a solid-like behavior.

The mechanical properties of erythrocytes have been investigated by different techniques. However, there are few reports on how the viscoelasticity of these cells varies during malaria disease. Here, we quantitatively map the viscoelastic properties of Plasmodium falciparum-parasitized human erythrocytes. We apply new methodologies based on optical tweezers to measure the viscoelastic properties and defocusing microscopy to measure the erythrocyte height profile, the overall cell volume, and its form factor, a crucial parameter to convert the complex elastic constant into complex shear modulus. The storage and loss shear moduli are obtained for each stage of parasite maturation inside red blood cells, while the former increase, the latter decrease. Employing a soft glassy rheology model, we obtain the power-law exponent for the storage and loss shear moduli, characterizing the soft glassy features of red blood cells in each parasite maturation stage. Ring forms present a liquid-like behavior, with a slightly lower power-law exponent than healthy erythrocytes, whereas trophozoite and schizont stages exhibit increasingly solid-like behaviors. Finally, the surface elastic shear moduli, low-frequency surface viscosities, and shape recovery relaxation times all increase not only in a stage-dependent manner but also when compared to healthy red blood cells. Overall, the results call attention to the soft glassy characteristics of Plasmodium falciparum-parasitized erythrocyte membrane and may provide a basis for future studies to better understand malaria disease from a mechanobiological perspective.

Host membrane glycosphingolipids and lipid microdomains facilitate Histoplasma capsulatum internalisation by macrophages.

Recognition and internalisation of intracellular pathogens by host cells is a multifactorial process, involving both stable and transient interactions. The plasticity of the host cell plasma membrane is fundamental in this infectious process. Here, the participation of macrophage lipid microdomains during adhesion and internalisation of the fungal pathogen Histoplasma capsulatum (Hc) was investigated. An increase in membrane lateral organisation, which is a characteristic of lipid microdomains, was observed during the first steps of Hc-macrophage interaction. Cholesterol enrichment in macrophage membranes around Hc contact regions and reduced levels of Hc-macrophage association after cholesterol removal also suggested the participation of lipid microdomains during Hc-macrophage interaction. Using optical tweezers to study cell-to-cell interactions, we showed that cholesterol depletion increased the time required for Hc adhesion. Additionally, fungal internalisation was significantly reduced under these conditions. Moreover, macrophages treated with the ceramide-glucosyltransferase inhibitor (P4r) and macrophages with altered ganglioside synthesis (from B4galnt1-/- mice) showed a deficient ability to interact with Hc. Coincubation of oligo-GM1 and treatment with Cholera toxin Subunit B, which recognises the ganglioside GM1, also reduced Hc association. Although purified GM1 did not alter Hc binding, treatment with P4 significantly increased the time required for Hc binding to macrophages. The content of CD18 was displaced from lipid microdomains in B4galnt1-/- macrophages. In addition, macrophages with reduced CD18 expression (CD18low ) were associated with Hc at levels similar to wild-type cells. Finally, CD11b and CD18 colocalised with GM1 during Hc-macrophage interaction. Our results indicate that lipid rafts and particularly complex gangliosides that reside in lipid rafts stabilise Hc-macrophage adhesion and mediate efficient internalisation during histoplasmosis.

Effects of cytoskeletal drugs on actin cortex elasticity.

Mechanical properties of cells are known to be influenced by the actin cytoskeleton. In this article, the action of drugs that interact with the actin cortex is investigated by tether extraction and rheology experiments using optical tweezers. The influences of Blebbistatin, Cytochalasin D and Jasplakinolide on the cell mechanical properties are evaluated. The results, in contradiction to current views for Jasplakinolide, show that all three drugs and treatments destabilize the actin cytoskeleton, decreasing the cell membrane tension. The cell membrane bending modulus increased when the actin cytoskeleton was disorganized by Cytochalasin D. This effect was not observed for Blebbistatin and Jasplakinolide. All drugs decreased by two-fold the cell viscoelastic moduli, but only Cytochalasin D was able to alter the actin network into a more fluid-like structure. The results can be interpreted as the interplay between the actin network and the distribution of myosins as actin cross-linkers in the cytoskeleton. This information may contribute to a better understanding of how the membrane and cytoskeleton are involved in cell mechanical properties, underlining the role that each one plays in these properties.

Antibody binding to Cryptococcus neoformans impairs budding by altering capsular mechanical properties.

Abs to microbial capsules are critical for host defense against encapsulated pathogens, but very little is known about the effects of Ab binding on the capsule, apart from producing qualitative capsular reactions ("quellung" effects). A problem in studying Ab-capsule interactions is the lack of experimental methodology, given that capsules are fragile, highly hydrated structures. In this study, we pioneered the use of optical tweezers microscopy to study Ab-capsule interactions. Binding of protective mAbs to the capsule of the fungal pathogen Cryptococcus neoformans impaired yeast budding by trapping newly emerging buds inside the parental capsule. This effect is due to profound mAb-mediated changes in capsular mechanical properties, demonstrated by a concentration-dependent increase in capsule stiffness. This increase involved mAb-mediated cross-linking of capsular polysaccharide molecules. These results provide new insights into Ab-mediated immunity, while suggesting a new nonclassical mechanism of Ab function, which may apply to other encapsulated pathogens. Our findings add to the growing body of evidence that Abs have direct antimicrobial functions independent of other components of the immune system.

Swimming behaviour of the multicellular magnetotactic prokaryote 'Candidatus Magnetoglobus multicellularis' under applied magnetic fields and ultraviolet light.

Magnetotactic bacteria move by rotating their flagella and concomitantly are aligned to magnetic fields because they present magnetosomes, which are intracellular organelles composed by membrane-bound magnetic crystals. This results in magnetotaxis, which is swimming along magnetic field lines. Magnetotactic bacteria are morphologically diverse, including cocci, rods, spirilla and multicellular forms known as magnetotactic multicellular prokaryotes (MMPs). 'Candidatus Magnetoglobus multicellularis' is presently the best known MMP. Here we describe the helical trajectories performed by these microorganisms as they swim forward, as well as their response to UV light. We measured the radius of the trajectory, time period and translational velocity (velocity along the helix axis), which enabled the calculation of other trajectory parameters such as pitch, tangential velocity (velocity along the helix path), angular frequency, and theta angle (the angle between the helix path and the helix axis). The data revealed that 'Ca. M. multicellularis' swims along elongated helical trajectories with diameters approaching the diameter of the microorganism. In addition, we observed that 'Ca. M. multicellularis' responds to UV laser pulses by swimming backwards, returning to forward swimming several seconds after the UV laser pulse. UV light from a fluorescence microscope showed a similar effect. Thus, phototaxis is used in addition to magnetotaxis in this microorganism.

Capsule of Cryptococcus neoformans grows by enlargement of polysaccharide molecules.

The human pathogenic fungus Cryptococcus neoformans has a distinctive polysaccharide (PS) capsule that enlarges during infection. The capsule is essential for virulence, but the mechanism for capsular growth is unknown. In the present study, we used dynamic light scattering (LS) analysis of capsular PS and optical tweezers (OT) to explore the architecture of the capsule. Analysis of capsular PS from cells with small and large capsules by dynamic LS revealed a linear correlation between PS effective diameter and microscopic capsular diameter. This result implied that capsule growth was achieved by the addition of molecules with larger effective diameter, such that some molecules can span the entire diameter of the capsule. Measurement of polystyrene bead penetration of C. neoformans capsules by using OT techniques revealed that the outer regions were penetrable, but not the inner regions. Our results provide a mechanism for capsular enlargement based on the axial lengthening of PS molecules and suggest a model for the architecture of a eukaryotic microbial capsule.

Quantitative Analysis of Viscoelastic Properties of Red Blood Cells using Optical Tweezers and Defocusing Microscopy.

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not only attributed to the normal variability of cells, but also to the differences in methods and models of cell response. Here, an integrated protocol using optical tweezers and defocusing microscopy is employed to obtain the rheological features of red blood cells in the frequency range of 1 Hz to 35 Hz. While optical tweezers are utilized to measure the erythrocyte-complex elastic constant, defocusing microscopy is able to obtain the cell height profile, volume, and its form factor a parameter that allows conversion of complex elastic constant into complex shear modulus. Moreover, applying a soft glassy rheology model, the scaling exponent for both moduli can be obtained. The developed methodology allows to explore the mechanical behavior of red blood cells, characterizing their viscoelastic parameters, obtained under well-defined experimental conditions, for several physiological and pathological conditions.

Chronological aging is associated with biophysical and chemical changes in the capsule of Cryptococcus neoformans.

Does the age of a microbial cell affect its virulence factors? To our knowledge, this question has not been addressed previously, but the answer is of great relevance for chronic infections where microbial cells persist and age in hosts. Cryptococcus neoformans is an encapsulated human-pathogenic fungus notorious for causing chronic infections where cells of variable age persist in tissue. The major virulence factor for C. neoformans is a polysaccharide (PS) capsule. To understand how chronological age could impact the cryptococcal capsule properties, we compared the elastic properties, permeabilities, zeta potentials, and glycosidic compositions of capsules from young and old cells and found significant differences in all parameters measured. Changes in capsular properties were paralleled by changes in PS molecular mass and density, as well as modified antigenic density and antiphagocytic properties. Remarkably, chronological aging under stationary-phase growth conditions was associated with the expression of α-1,3-glucans in the capsule, indicating a new structural capsular component. Our results establish that cryptococcal capsules are highly dynamic structures that change dramatically with chronological aging under prolonged stationary-phase growth conditions. Changes associated with cellular aging in chronic infections could contribute to the remarkable capacity of this fungus to persist in tissues by generating phenotypically and antigenically different capsules.

Thyroid hormone induces cerebellar neuronal migration and Bergmann glia differentiation through epidermal growth factor/mitogen-activated protein kinase pathway.

Cerebellar development in the postnatal period is mainly characterized by an intense cellular proliferation in the external granular layer, followed by migration of granular cells in the molecular layer along the Bergmann glia (BG) fibers. Cerebellar ontogenesis undergoes dramatic modulation by thyroid hormones (THs), although their mechanism of action in this organ is still largely unknown. We previously demonstrated that THs induce astrocytes to secrete epidermal growth factor (EGF), which thus promotes cerebellar neuronal proliferation and extracellular matrix remodeling in vitro. In the present study, we investigated the effect of the TH/EGF pathway on granule neuronal migration. By taking advantage of rat explant and dissociated culture assays, we showed that cerebellar astrocytes treated with TH promote granule cell migration. The addition of neutralizing antibodies against EGF or the pharmacological inhibitor of EGF signaling, bis-tyrphostin, completely inhibited TH-astrocyte-induced migration. Likewise, the addition of EGF itself greatly increased neuronal migration. Treatment of BG-dissociated cultures by EGF dramatically induced an alteration in cell morphology, characterized by an elongation in the glial process. Both neuronal migration and BG elongation were inhibited by the mitogen-activated protein kinase pathway inhibitor PD98059, suggesting that these events might be associated. Together, our results suggest that, by inducing EGF secretion, THs promote neuronal migration through BG elongation. Our data provide new clues to the molecular mechanism of THs in cerebellar development, and may contribute to a better understanding of some neuroendocrine disorders associated with migration deficits.

CTGF/CCN2 has a chemoattractive function but a weak adhesive property to embryonic carcinoma cells.

Connective tissue growth factor (CTGF/CCN2) is a protein of the CCN family that modulates cell-ECM interactions in a variety of cell types. In this study, we investigated the chemotactic and adhesive properties of CCN2 protein in embryonic teratocarcinoma P19 cells. Initially, P19 cells were attracted to CCN2-coated agarose beads. In Boyden chamber experiments, CCN2-containing medium induced a threefold greater migration of P19 cells. CCN2 adhesion properties were studied by using optical tweezers. The specific adhesion times of P19 cells to polystyrene beads coated with laminin, fibronectin, CCN2 and bovine serum albumin were 1.8 ± 0.5s, 2.7 ± 0.4s, 10 ± 2s and 13 ± 2s, respectively, revealing an unexpectedly low adhesive capacity of CCN2 protein for P19 cells. In conclusion, our findings support the chemoattractive role of CCN2 for P19 cells, but not its adhesive role when compared to laminin or fibronectin.

Protocol to measure the membrane tension and bending modulus of cells using optical tweezers and scanning electron microscopy.

The elastic properties of cell membranes, particularly the membrane tension and bending modulus, are known to be key regulators of cellular functions. Here, we present a correlative and integrated tool based on optical tweezers and scanning electron microscopy to accurately determine these properties in a variety of cell types. Although there are intrinsic difficulties associated with correlative experiments, we believe that the methods presented can be considered a suitable protocol for determining the elastic properties of cell membranes. For complete details on the use and execution of this protocol, please refer to Soares et al. (2020).

Membrane Elastic Properties During Neural Precursor Cell Differentiation.

Neural precursor cells differentiate into several cell types that display distinct functions. However, little is known about how cell surface mechanics vary during the differentiation process. Here, by precisely measuring membrane tension and bending modulus, we map their variations and correlate them with changes in neural precursor cell morphology along their distinct differentiation fates. Both cells maintained in culture as neural precursors as well as those plated in neurobasal medium reveal a decrease in membrane tension over the first hours of culture followed by stabilization, with no change in bending modulus. During astrocyte differentiation, membrane tension initially decreases and then increases after 72 h, accompanied by consolidation of glial fibrillary acidic protein expression and striking actin reorganization, while bending modulus increases following observed alterations. For oligodendrocytes, the changes in membrane tension are less abrupt over the first hours, but their values subsequently decrease, correlating with a shift from oligodendrocyte marker O4 to myelin basic protein expressions and a remarkable actin reorganization, while bending modulus remains constant. Oligodendrocytes at later differentiation stages show membrane vesicles with similar membrane tension but higher bending modulus as compared to the cell surface. Altogether, our results display an entire spectrum of how membrane elastic properties are varying, thus contributing to a better understanding of neural differentiation from a mechanobiological perspective.

The elastic properties of the Cryptococcus neoformans capsule.

Microbial capsules are important for virulence, but their architecture and physical properties are poorly understood. The human pathogenic fungus Cryptococcus neoformans has a large polysaccharide capsule that is necessary for virulence and is the target of protective antibody responses. To study the C. neoformans capsule we developed what we believe is a new approach whereby we probed the capsular elastic properties by applying forces using polystyrene beads manipulated with optical tweezers. This method allowed us to determine the Young's modulus for the capsule in various conditions that affect capsule growth. The results indicate that the Young's modulus of the capsule decreases with its size and increases with the Ca(2+) concentration in solution. Also, capsular polysaccharide manifests an unexpected affinity for polystyrene beads, a property that may function in attachment to host cells and environmental structures. Bead probing with optical tweezers provides a new, nondestructive method that may have wide applicability for studying the effects of growth conditions, immune components, and drugs on capsular properties.

Cryptococcus neoformans capsular polysaccharide and exopolysaccharide fractions manifest physical, chemical, and antigenic differences.

The human pathogenic fungus Cryptococcus neoformans has a large polysaccharide (PS) capsule and releases copious amounts of PS into cultures and infected tissues. The capsular PS is a major virulence factor that can elicit protective antibody responses. PS recovered from culture supernatants has historically provided an ample and convenient source of material for structural and immunological studies. Two major assumptions in such studies are that the structural features of the exopolysaccharide material faithfully mirror those of capsular PS and that the isolation methods do not change PS properties. However, a comparison of exopolysaccharide made by two isolation techniques with capsular PS stripped from cells with gamma radiation or dimethyl sulfoxide revealed significant differences in glycosyl composition, mass, size, charge, viscosity, circular-dichroism spectra, and reactivity with monoclonal antibodies. Our results strongly suggest that exopolysaccharides and capsular PS are structurally different. A noteworthy finding was that PS made by cetyltrimethylammonium bromide precipitation had a larger mass and a different conformation than PS isolated by concentration and filtration, suggesting that the method most commonly used to purify glucuronoxylomannan alters the PS. Hence, the method used to isolate PS can significantly influence the structural and antigenic properties of the product. Our findings have important implications for current views of the relationship between capsular PS and exopolysaccharides, for the generation of PS preparations suitable for immunological studies, and for the formulation of PS-based vaccines for the prevention of cryptococcosis.

The mechanical properties of microbial surfaces and biofilms.

Microbes can modify their surface structure as an adaptive mechanism for survival and dissemination in the environment or inside the host. Altering their ability to respond to mechanical stimuli is part of this adaptive process. Since the 1990s, powerful micromanipulation tools have been developed that allow mechanical studies of microbial cell surfaces, exploring little known aspects of their dynamic behavior. This review concentrates on the study of mechanical and rheological properties of bacteria and fungi, focusing on their cell surface dynamics and biofilm formation.

Rheological properties of cryptococcal polysaccharide change with fiber size, antibody binding and temperature.

Aim:Cryptococcus neoformans is the major agent of cryptococcosis. The main virulence factor is the polysaccharide (PS) capsule. Changes in cryptococcal PS properties have been poorly elucidated. Materials & methods: We analyzed the mechanical properties of secreted PS and intact capsules, using dynamic light scattering and optical tweezers. Results: Storage and loss moduli showed that secreted PS behaves as a viscoelastic liquid, while capsular PS behaves as a viscoelastic solid. The secreted PS remains as a viscoelastic fluid at different temperatures with thermal hysteresis after 85°C. Antibody binding altered the viscoelastic behavior of both secreted and capsular PS. Conclusion: Deciphering the mechanical aspects of these structures could reveal features that may have consequences in novel therapies against cryptococcosis.

Exact Theory of Optical Tweezers and Its Application to Absolute Calibration.

Optical tweezers have become a powerful tool for basic and applied research in cell biology. Here, we describe an experimentally verified theory for the trapping forces generated by optical tweezers based on first principles that allows absolute calibration. For pedagogical reasons, the steps that led to the development of the theory over the past 15 years are outlined. The results are applicable to a broad range of microsphere radii, from the Rayleigh regime to the ray optics one, for different polarizations and trapping heights, including all commonly employed parameter domains. Protocols for implementing absolute calibration are given, explaining how to measure all required experimental parameters, and including a link to an applet for stiffness calculations.

Rheological properties of cells measured by optical tweezers.

BACKGROUND: The viscoelastic properties of cells have been investigated by a variety of techniques. However, the experimental data reported in literature for viscoelastic moduli differ by up to three orders of magnitude. This has been attributed to differences in techniques and models for cell response as well as to the natural variability of cells. RESULTS: In this work we develop and apply a new methodology based on optical tweezers to investigate the rheological behavior of fibroblasts, neurons and astrocytes in the frequency range from 1Hz to 35Hz, determining the storage and loss moduli of their membrane-cortex complex. To avoid distortions associated with cell probing techniques, we use a previously developed method that takes into account the influence of under bead cell thickness and bead immersion. These two parameters were carefully measured for the three cell types used. Employing the soft glass rheology model, we obtain the scaling exponent and the Young's modulus for each cell type. The obtained viscoelastic moduli are in the order of Pa. Among the three cell types, astrocytes have the lowest elastic modulus, while neurons and fibroblasts exhibit a more solid-like behavior. CONCLUSIONS: Although some discrepancies with previous results remain and may be inevitable in view of natural variability, the methodology developed in this work allows us to explore the viscoelastic behavior of the membrane-cortex complex of different cell types as well as to compare their viscous and elastic moduli, obtained under identical and well-defined experimental conditions, relating them to the cell functions.

Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions.

Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM) of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D) environment mainly composed of Collagen I (COL I). This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited "freeze and run" migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular "home"-macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model.

Membrane elastic properties and cell function.

Recent studies indicate that the cell membrane, interacting with its attached cytoskeleton, is an important regulator of cell function, exerting and responding to forces. We investigate this relationship by looking for connections between cell membrane elastic properties, especially surface tension and bending modulus, and cell function. Those properties are measured by pulling tethers from the cell membrane with optical tweezers. Their values are determined for all major cell types of the central nervous system, as well as for macrophage. Astrocytes and glioblastoma cells, which are considerably more dynamic than neurons, have substantially larger surface tensions. Resting microglia, which continually scan their environment through motility and protrusions, have the highest elastic constants, with values similar to those for resting macrophage. For both microglia and macrophage, we find a sharp softening of bending modulus between their resting and activated forms, which is very advantageous for their acquisition of phagocytic functions upon activation. We also determine the elastic constants of pure cell membrane, with no attached cytoskeleton. For all cell types, the presence of F-actin within tethers, contrary to conventional wisdom, is confirmed. Our findings suggest the existence of a close connection between membrane elastic constants and cell function.