Candida albicans and mutans streptococci are frequently detected in dental plaque biofilms from toddlers afflicted with early childhood caries. Glucosyltransferases (Gtfs) secreted by Streptococcus mutans bind to saliva-coated apatite (sHA) and to bacterial surfaces, synthesizing exopolymers in situ, which promote cell clustering and adherence to tooth enamel. We investigated the potential role Gtfs may play in mediating the interactions between C. albicans SC5314 and S. mutans UA159, both with each other and with the sHA surface. GtfB adhered effectively to the C. albicans yeast cell surface in an enzymatically active form, as determined by scintillation spectroscopy and fluorescence imaging. The glucans formed on the yeast cell surface were more susceptible to dextranase than those synthesized in solution or on sHA and bacterial cell surfaces (P < 0.05), indicating an elevated ␣-1,6-linked glucose content. Fluorescence imaging revealed that larger numbers of S. mutans cells bound to C. albicans cells with glucans present on their surface than to yeast cells without surface glucans (uncoated). The glucans formed in situ also enhanced C. albicans interactions with sHA, as determined by a novel single-cell micromechanical method. Furthermore, the presence of glucan-coated yeast cells significantly increased the accumulation of S. mutans on the sHA surface (versus S. mutans incubated alone or mixed with uncoated C. albicans; P < 0.05). These data reveal a novel cross-kingdom interaction that is mediated by bacterial GtfB, which readily attaches to the yeast cell surface. Surface-bound GtfB promotes the formation of a glucan-rich matrix in situ and may enhance the accumulation of S. mutans on the tooth enamel surface, thereby modulating the development of virulent biofilms.

Bilayer membranes are intrinsically fluid in character and require stabilization by association with an underlying cytoskeleton. Instability either in the membrane-associated cytoskeleton or in the association between the bilayer and the skeleton can lead to loss of membrane bilayer and premature cell death. In this report measurements of the physical strength of the association between membrane bilayer and the membrane-associated skeleton in red blood cells are reported. These measurements involve the mechanical formation of long, thin cylinders of membrane bilayer (tethers) from the red cell surface. Ultrastructural evidence is presented indicating that these tethers do not contain membrane skeleton and, furthermore, that they are deficient in at least some integral membrane proteins. By measuring the forces on the cell as the tether is formed and the dimensions of the tether, the energy associated with its formation can be calculated. The minimum force to form a tether was found to be approximately 50 pN corresponding to an energy of dissociation of 0.2-0.3 mJ/m2. Such measurements enable critical evaluation of potential physical mechanisms for the stabilization of the membrane bilayer by the underlying cytoskeleton. It is postulated that an important contribution to the energy of association between bilayer and skeleton comes from the increase in chemical potential due to the lateral segregation of lipids and integral proteins.

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During maturation of the red blood cell (RBC) from the nucleated normoblast stage to the mature biconcave discocyte, both the structure and mechanical properties of the cell undergo radical changes. The development of the mechanical stability of the membrane reflects underlying changes in the organization of membraneassociated cytoskeletal proteins, and so provides an assessment of the time course of the development of membrane structural organization. Membrane stability in maturing erythrocytes was assessed by measuring forces required to form thin, tubular, lipid strands (tethers) from the surfaces of mononuclear cells obtained from fresh human marrow samples, marrow reticulocytes, circulating reticulocytes, and mature erythrocytes. Cells were biotinylated and manipulated with a micropipette to form an adhesive contact with a glass microcantilever, which gave a measure of the tethering force. The cell was withdrawn at controlled velocity and aspiration pressure to form a tether from the cell surface. The mean force required to form tethers from marrow reticulocytes and normoblasts was 27 ؎ 9 pN, compared to 54 ؎ 14 pN for mature cells. The energy of dissociation of the bilayer from the underlying skeleton increases 4-fold between the marrow reticulocyte stage and the mature cell, demonstrating that the mechanical stability of the membrane is not completely established until the very last stages of RBC maturation. IntroductionDuring the last stages of maturation of red blood cells (RBCs), dramatic changes occur in the structure and organization within the cell. The cell loses its nucleus, surface molecules are shed in small vesicles, and the final surface-to-volume ratio of the cell is established. [1][2][3] During this time, proteins that will eventually form the membrane-associated cytoskeleton (membrane skeleton) are synthesized and assembled at the intracellular surface of the plasma membrane. 4 The time course over which these protein assemblies become functionally viable is of interest, particularly with regard to hemolytic anemia and the early release of cells during hemorrhagic crisis, and could be important in designing methods for production of erythrocytes in vitro.The function of the assembled membrane skeleton is fundamentally mechanical, and therefore, studies of membrane mechanical properties in maturing cells provide the most direct assessment of the development of the functional viability of the skeleton during maturation. Early studies of membrane properties of both murine and human reticulocytes indicated increased membrane stiffness (shear rigidity) in those membranes. 5 This increased rigidity has been confirmed subsequently both by micropipette 6 and cell deformation in shear (ektacytometry). 7 In the latter study, evidence was also obtained that, despite increased mechanical stiffness, membranes of immature cells were less mechanically stable than their mature counterparts, as indicated by fragmentation of cells in fluid shear and in micropipette aspiration studies. The structural events...

During murine embryogenesis, primitive erythroblasts enter the circulation as immature nucleated cells and progressively mature as a semisynchronous cohort, enucleating between E12.5 and E16.5. In this report, we examine the mechanical properties of these cells to determine how their mechanical development differs from that of definitive erythroid cells, which mature extravascularly in protected marrow microenvironments. Primitive erythroid cells acquire normal membrane deformability by E12.5 (i.e., as late stage erythroblasts) and maintain the same level of surface stiffness through E17.5. During this same period, the strength of association between the membrane bilayer and the underlying skeleton increases, as indicated by an approximate doubling of the energy required to separate bilayer from skeleton. At the same time, these cells undergo dramatic changes in surface area and volume, losing 35% of their surface area and 50% of their volume from E14.5 to E17.5. Interestingly, membrane remodeling proceeded regardless of whether the cells completed enucleation. These data suggest that in primitive erythroid cells, unlike their definitive counterparts, the critical maturational processes of membrane remodeling and enucleation are uncoupled.