Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm−1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s−1 under zero force up to 15 s−1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

Controlled unzipping of a bacterial surface layer with atomic force microscopy

We have combined high-resolution atomic force microscopy (AFM) imaging and force spectroscopy to gain insight into the interaction forces between the individual protomers of the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans. After imaging the HPI layer, the AFM stylus was attached to individual protomers by enforced stylus-sample contact to allow force spectroscopy experiments. Imaging of the HPI layer after recording force-extension curves allowed adhesion forces to be correlated with structural alterations. By using this approach, individual protomers of the HPI layer were found to be removed at pulling forces of ≈300 pN. Furthermore, it was possible to sequentially unzip entire bacterial pores formed by six HPI protomers. The combination of high-resolution AFM imaging of individual proteins with the determination of their intramolecular forces is a method of studying the mechanical stability of supramolecular structures at the level of single molecules.

Stretching single-domain proteins: Phase diagram and kinetics of force-induced unfolding

Single-molecule force spectroscopy reveals unfolding of domains in titin on stretching. We provide a theoretical framework for these experiments by computing the phase diagrams for force-induced unfolding of single-domain proteins using lattice models. The results show that two-state folders (at zero force) unravel cooperatively, whereas stretching of non-two-state folders occurs through intermediates. The stretching rates of individual molecules show great variations reflecting the heterogeneity of force-induced unfolding pathways. The approach to the stretched state occurs in a stepwise “quantized” manner. Unfolding dynamics and forces required to stretch proteins depend sensitively on topology. The unfolding rates increase exponentially with force f till an optimum value, which is determined by the barrier to unfolding when f = 0. A mapping of these results to proteins shows qualitative agreement with force-induced unfolding of Ig-like domains in titin. We show that single-molecule force spectroscopy can be used to map the folding free energy landscape of proteins in the absence of denaturants.

Unbinding process of adsorbed proteins under external stress studied by atomic force microscopy spectroscopy

We report the study of the dynamics of the unbinding process under a force load f of adsorbed proteins (fibrinogen) on a solid surface (hydrophilic silica) by means of atomic force microscopy spectroscopy. By varying the loading rate rf, defined by f = rf t, t being the time, we find that, as for specific interactions, the mean rupture force increases with rf. This unbinding process is analyzed in the framework of the widely used Bell model. The typical dissociation rate at zero force entering in the model lies between 0.02 and 0.6 s−1. Each measured rupture is characterized by a force f0, which appears to be quantized in integer multiples of 180–200 pN.

Static and dynamic lengths of neutrophil microvilli

Containing most of the L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) on their tips, microvilli are believed to promote the initial arrest of neutrophils on endothelium. At the rolling stage following arrest, the lifetimes of the involved molecular bonds depend on the pulling force imposed by the shear stress of blood flow. With two different methods, electron microscopy and micropipette manipulation, we have obtained two comparable neutrophil microvillus lengths, both ≈0.3 μm in average. We have found also that, under a pulling force, a microvillus can be extended (microvillus extension) or a long thin membrane cylinder (a tether) can be formed from it (tether formation). If the force is ≤34 pN (± 3 pN), the length of the microvillus will be extended; if the force is >61 pN (± 5 pN), a tether will be formed from the microvillus at a constant velocity, which depends linearly on the force. When the force is between 34 pN and 61 pN (transition zone), the degree of association between membrane and cytoskeleton in individual microvilli will dictate whether microvillus extension or tether formation occurs. When a microvillus is extended, it acts like a spring with a spring constant of ≈43 pN/μm. In contrast to a rigid or nonextendible microvillus, both microvillus extension and tether formation can decrease the pulling force imposed on the adhesive bonds, and thus prolonging the persistence of the bonds at high physiological shear stresses.

Dynamics of Centromeres during Metaphase–Anaphase Transition in Fission Yeast: Dis1 Is Implicated in Force Balance in Metaphase Bipolar Spindle

In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.

Direct measurement of hydrogen bonding in DNA nucleotide bases by atomic force microscopy.

We have used self-assembled purines and pyrimidines on planar gold surfaces and on gold-coated atomic force microscope (AFM) tips to directly probe intermolecular hydrogen bonds. Electron spectroscopy for chemical analysis (ESCA) and thermal programmed desorption (TPD) measurements of the molecular layers suggested monolayer coverage and a desorption energy of about 25 kcal/mol. Experiments were performed under water, with all four DNA bases immobilized on AFM tips and flat surfaces. Directional hydrogen-bonding interaction between the tip molecules and the surface molecules could be measured only when opposite base-pair coatings were used. The directional interactions were inhibited by excess nucleotide base in solution. Nondirectional van der Waals forces were present in all other cases. Forces as low as two interacting base pairs have been measured. With coated AFM tips, surface chemistry-sensitive recognition atomic force microscopy can be performed.