The force regulation on binding kinetics and conformations of integrin and selectins using a bio-membrane force probe.

摘要

Cell adhesion plays an important role in inflammation and immunological responses. Adhesion molecules (e.g., selectins and integrins) are key modulators in mediating these cellular responses. The central theme of this thesis is to investigate the relation between force and kinetics rates and to dissect its mechanism on molecular basis.;In order to study the relation between force, kinetic rates and structural conformational changes, an advanced single molecule instrument, the bio-membrane force probe (BFP), was implemented to explore these fundamental biological questions. This instrument allowed us to measure force-dependent lifetimes of individual molecular bonds with a force sensitivity of ∼1 pN and to detect molecular conformational changes larger than ∼5 nm. We developed a new thermal fluctuation assay to monitor P-, L-selectin and PSGL-1 interactions between two opposing surfaces and to estimate their 2D on-rate and off-rates simultaneously.;Using this new BFP, the relation between force and force history of selectin-ligand interactions was investigated. Based on the sliding rebinding model, the flexibility of L-selectin is an important factor regulating selectin's catch bonds. The lifetimes of bonds between the L-selectin hinge domain mutant (L-selectinN138G) and ligands were measured. The lifetime results showed that increasing the flexibility of the lectin domain by eliminating a hydrogen bond in the inter-domain hinge augmented the bond lifetimes in catch bond regimes. More interestingly, one more slip bond regime was observed in all three selectins at lower forces where previous experiments had never measured. All three selectins exhibited tri-phasic transition of force-dependent off-rates in interacting with their ligands. This tri-phasic transition could further support the sliding rebinding model as this model had predicted that breaking the interactions between the lectin and EGF domains before the lectin domain's pivoting could result in a typical slip bond, as postulated by G. Bell. In addition to regulation of catch bonds by biochemical factors, our results showed that biophysical factors (e.g., force loading rates) could also change the catch bonds' behavior. That is, as loading rates increased, L-selectin-PSGL-1's catch bonds gradually shifted to lower force and were completely converted to slip bonds once force loading rate increased to 104 pN/s. However, catch bonds in L-selectin-6-sulfo-sLex interactions were not changed by these increased loading rates. The mechanism of force loading rating regulation on catch bonds still remains elusive. More deep analysis and experiments are needed to reveal the mechanism.;The BFP was also used to characterize force-dependent lifetimes of LFA-1-ICAM-1 interactions. It has been hypothesized that catch bonds might also exist in LFA-1-ICAM-1 bonds due to the multiple affinity states of LFA-1's ligand binding domain (alphaA domain). The experiments on the bond lifetimes between ICAM-1 and LFA-1 expressed on Jurkat cells showed that LFA-1/ICAM-1 bonds indeed behaved as catch bonds. As a control, the lifetimes of LFA-1 and anti-LFA-1 mAb (KIM127) showed slip bonds. Lifetime distributions of catch bonds revealed a two-state transition, while those of slip bonds did not. More importantly, LFA-1-ICAM-1's catch bonds were abolished by the binding of the small allosteric molecule, XVA143, to LFA-1 and converted to slip bonds. This abolishment provided a structural mechanism for catch bonds in LFA-1-ICAM-1 interactions, that is, the downward movement of alpha7 helix in alphaA domain adjusted the MIDAS in alphaA domain to a high affinity state by mechanical forces; such movement may prolong bond lifetimes in lower force regimes.;Finally, the BFP was applied to dynamically probe the global conformational changes of LFA-1 and to characterize force-regulated transitions among different conformational states on a living cell. Our results showed that dynamic transitions of LFA-1 between extended and bent conformations were probed on living cells by the measurement of force-independent distance changes of the BFP probe beads, by examining the frequencies with which these changes occur (they were modulated by cations, small allosteric molecule, XVA143, and different antibodies (i.e., KIM127 and MEM83)), and by mechanical analysis of LFA-1-ICAM-1 complexes in the above conditions. The observed average distance change of LFA-1's extensions was about 18nm, while that of the contraction was only about 14nm. Finally, our results showed that forces could facilitate extension but they slow down contraction of LFA-1. The observed transition time of extension was =0.1s, while that of contraction was >0.2s. (Abstract shortened by UMI.)