Phagocytes clear the body of undesirable particles such as infectious agents and debris. To extend pseudopods over the surface of targeted particles during engulfment, cells must change shape through extensive membrane and cytoskeleton remodeling. We observed that pseudopod extension occurred in two phases. In the first phase, pseudopods extended rapidly, with actin polymerization pushing the plasma membrane forward. The second phase occurred once the membrane area from preexisting reservoirs was depleted, leading to increased membrane tension. Increased tension directly altered the small Rho GTPase Rac1, 3′-phosphoinositide, and cytoskeletal organization. Furthermore, it activated exocytosis of vesicles containing GPI-anchored proteins, increasing membrane area and phagocytosis efficiency for large particles. We thus propose that, during phagocytosis, membrane remodeling, cytoskeletal organization, and biochemical signaling are orchestrated by the mechanical signal of membrane tension. These results put a simple mechanical signal at the heart of understanding immunological responses.

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.

No abstract

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.Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis, particularly in immunocompromised patients. The fungus is unique among human eukaryotic pathogens in that it has a polysaccharide (PS) capsule that is the major determinant of virulence (1). The capsule is composed of 2 major PS, galactoxylomannan (GalXM) and glucuronoxylomannan (GXM). GalXM is an ␣(1, 6)-galactan containing ␤(1, 3)oligosaccharyl substitutions at alternate residues of galactose. The oligosaccharide constituents of GalXM are trisaccharide motifs composed of ␣(1, 3)-mannosyl dimers in ␣ (1, 4) linkages to galatosyl units. Each of the trisaccharide components may be substituted with ␤(1, 2)-or ␤(1, 3)-glucuronyl residues. GXM consists of a ␣-(1, 3)-mannan main chain with ␤(1, 2)-glucuronic acid residues attached to every third mannose, on average. Mannosyl residues can also be 6-O-acetylated and substituted with xylosyl units in ␤(1, 2)-or ␤(1, 4)-linkages (2). GXM and GalXM are released into culture media by growing cells as exo-PS that can be recovered for physical and chemical analysis. When coupled to protein, GXM can elicit protective antibodies (3).C. neoformans cells increase their capsular diameter in response to diverse stressors, including mammalian infection (4). Capsule enlargement has been associated with virulence (5-8), and it protects the fungus against host defense mechanisms, such as phagocytosis and oxidative burst (9, 10). Although the biological properties of the capsule have been extensively studied, its architecture and mechanism of enlargement have not been fully elucidated. Capsule enlargement can occur by apical growth (7), and there is evidence that capsule size is regulated at the level of individual PS molecules (11). Given that GXM is a macromolecule, and that capsular assembly involves the noncovalent attachment of PS fibrils to the cell wall (12), and to each other (13, 14), it is likely that many aspects of capsule ...

We perform a detailed investigation of the force × deformation curve in tether extraction from 3T3 cells by optical tweezers. Contrary to conventional wisdom about tethers extracted from cells, we find that actin filaments are present within them, so that a revised theory of tether pulling from cells is called for. We also measure steady and maximum tether force values significantly higher than previously published ones for 3T3 cells. Possible explanations for these differences are investigated. Further experimental support of the theory of force barriers for membrane tube extension is obtained. The potential of studies on tether pulling force × deformation for retrieving information on membrane-cytoskeleton interaction is emphasized.

Pontes et al. show that plasma membrane mechanics exerts an upstream control during cell motility. Variations in plasma membrane tension orchestrate the behavior of the cell leading edge, with increase–decrease cycles in tension promoting adhesion row positioning.

We propose to use optical tweezers to probe the Casimir interaction between microspheres inside a liquid medium for geometric aspect ratios far beyond the validity of the widely employed proximity force approximation. This setup has the potential for revealing unprecedented features associated to the non-trivial role of the spherical curvatures. For a proof of concept, we measure femtonewton double layer forces between polystyrene microspheres at distances above 400 nm by employing very soft optical tweezers, with stiffness of the order of fractions of a fN/nm. As a future application, we propose to tune the Casimir interaction between a metallic and a polystyrene microsphere in saline solution from attraction to repulsion by varying the salt concentration. With those materials, the screened Casimir interaction may have a larger magnitude than the unscreened one. This line of investigation has the potential for bringing together different fields including classical and quantum optics, statistical physics and colloid science, while paving the way for novel quantitative applications of optical tweezers in cell and molecular biology.

Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular structures in pathogenic Cryptococcus species and environmental species share similar features, but also manifest significant difference that could influence their potential to virulence.