Tyrosine replacement in P-selectin glycoprotein ligand-1 affects distinct kinetic and mechanical properties of bonds with P- and L-selectin

Selectins are adhesion molecules that initiate tethering and rolling of leukocytes on the vessel wall. Rolling requires rapid formation and breakage of selectin–ligand bonds that must have mechanical strength to resist premature dissociation by the forces applied in shear flow. P- and L-selectin bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. To define determinants on PSGL-1 that contribute to the kinetic and mechanical properties of bonds with selectins, we compared rolling of transfected preB cells expressing P- or L-selectin on transfected cell monolayers expressing wild-type PSGL-1 or PSGL-1 constructs with substitutions in targeted N-terminal residues. Rolling through P- or L-selectin required a Thr or Ser at a specific position on PSGL-1, the attachment site for an essential O-glycan, but required only one of three nearby Tyr residues, which are sites for Tyr-SO3 formation. The adhesive strengths and numbers of cells rolling through P- or L-selectin were similar on wild-type PSGL-1 and on each of the three PSGL-1 constructs containing only a single Tyr. However, the cells rolled more irregularly on the single-Tyr forms of PSGL-1. Analysis of the lifetimes of transient tethers on limiting densities of PSGL-1 revealed that L-selectin dissociated faster from single-Tyr than wild-type PSGL-1 at all shears examined. In sharp contrast, P-selectin dissociated faster from single-Tyr than wild-type PSGL-1 at higher shear but not at lower shear. Thus, tyrosine replacements in PSGL-1 affect distinct kinetic and mechanical properties of bonds with P- and L-selectin.

The mechanical properties of Saccharomyces cerevisiae

Cell-wall mechanical properties play an integral part in the growth and form of Saccharomyces cerevisiae. In contrast to the tremendous knowledge on the genetics of S. cerevisiae, almost nothing is known about its mechanical properties. We have developed a micromanipulation technique to measure the force required to burst single cells and have recently established a mathematical model to extract the mechanical properties of the cell wall from such data. Here we determine the average surface modulus of the S. cerevisiae cell wall to be 11.1 ± 0.6 N/m and 12.9 ± 0.7 N/m in exponential and stationary phases, respectively, giving corresponding Young's moduli of 112 ± 6 MPa and 107 ± 6 MPa. This result demonstrates that yeast cell populations strengthen as they enter stationary phase by increasing wall thickness and hence the surface modulus, without altering the average elastic properties of the cell-wall material. We also determined the average breaking strain of the cell wall to be 82% ± 3% in exponential phase and 80% ± 3% in stationary phase. This finding provides a failure criterion that can be used to predict when applied stresses (e.g., because of fluid flow) will lead to wall rupture. This work analyzes yeast compression experiments in different growth phases by using engineering methodology.