Measuring Molecular Elasticity by Atomic Force Microscope Cantilever Fluctuations

Marshall, Bryan T.; Sarangapani, Krishna K.; Wu, Jianhua; Lawrence, Michael B.; McEver, Rodger P.; Zhu, Cheng

doi:10.1529/biophysj.105.061010

Cited by 75 publications

(59 citation statements)

References 42 publications

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“…We have previously used AFM for mechanical measurements of L-and P-selectin (10). Here, we expand the previous study by systematically characterizing the dead zone and stiffness of all three selectins complexed with different ligands and mAbs.…”

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confidence: 73%

Molecular Stiffness of Selectins

Sarangapani¹,

Marshall

McEver

et al. 2011

Journal of Biological Chemistry

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During inflammation, selectin-ligand interactions provide forces for circulating leukocytes to adhere to vascular surfaces, which stretch the interacting molecules, suggesting that mechanical properties may be pertinent to their biological function. From mechanical measurements with atomic force microscopy, we analyzed the molecular characteristics of selectins complexed with ligands and antibodies. Respective stiffness of L-, E-, and P-selectins (4.2, 1.4, and 0.85 piconewton/nm) correlated inversely with the number (2, 6, and 9) of consensus repeats in the selectin structures that acted as springs in series to dominate their compliance. After reconstitution into a lipid bilayer, purified membrane P-selectin remained a dimer, capable of forming dimeric bonds with P-selectin glycoprotein ligand (PSGL)-1, endoglycan-Ig, and a dimeric form of a glycosulfopeptide modeled after the N terminus of PSGL-1. By comparison, purified membrane L-and E-selectin formed only monomeric bonds under identical conditions. Ligands and antibodies were much less stretchable than selectins. The length of endoglycan-Ig was found to be 51 ؎ 12 nm. These results provide a comprehensive characterization of the molecular stiffness of selectins and illustrate how mechanical measurements can be utilized for molecular analysis, e.g. evaluating the multimericity of selectins and determining the molecular length of endoglycan.The physical properties of biomolecules can be exploited as tools for their analysis. For instance, electrophoresis that separates proteins according to mass and charge can be exploited to analyze molecular identity and abundance. Physical properties of many biomolecules also have critical roles in their functions. As an example, bending rigidities of actin and microtubule enable these cytoskeletal proteins to provide mechanical support to the cell. Adhesion molecules are also subjected to forces because they anchor cells to other cells or to the extracellular matrix. Mechanical properties and their relevance to biological function of DNA and muscle proteins (e.g. titin and ubiquitin) have been extensively documented (1-8). However, limited studies exist on the mechanical characterization of adhesion molecules and on the utilization of such mechanical measurements for molecular analysis. Here, we employ atomic force microscopy (AFM) 4 to measure mechanical properties of molecular complexes of selectins and their ligands or monoclonal antibodies (mAbs) and use mechanical measurements to address biological issues involving these molecules.Each of the three selectins consists of an N-terminal lectin domain, an epidermal growth factor (EGF)-like domain, 2, 6, or 9 (for L-, E-or P-selectin, respectively) short consensus repeats (CRs), a transmembrane domain, and a short cytoplasmic tail (Fig. 1A). During inflammation, selectin-ligand interactions mediate tethering and rolling of circulating leukocytes on vascular surfaces under a mechanically stressful milieu (9). These molecular complexes are subjected to physical forces that ...

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confidence: 73%

Molecular Stiffness of Selectins

Sarangapani¹,

Marshall

McEver

et al. 2011

Journal of Biological Chemistry

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“…S1) or nonspecific binding controls. A TFZ has been observed in interactions between lengthy molecules because they need to be fully extended before they can resist any tensile forces (26,27). In G-actin/F-actin interactions, the TFZ is interpreted as a period (and length of displacement) during which the G-actin/F-actin bond is formed but the F-actin filament is not fully extended (Fig.…”

Section: Resultsmentioning

confidence: 99%

Actin depolymerization under force is governed by lysine 113:glutamic acid 195-mediated catch-slip bonds

Lee

Lou

Wen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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As a key element in the cytoskeleton, actin filaments are highly dynamic structures that constantly sustain forces. However, the fundamental question of how force regulates actin dynamics is unclear. Using atomic force microscopy force-clamp experiments, we show that tensile force regulates G-actin/G-actin and G-actin/F-actin dissociation kinetics by prolonging bond lifetimes (catch bonds) at a low force range and by shortening bond lifetimes (slip bonds) beyond a threshold. Steered molecular dynamics simulations reveal force-induced formation of new interactions that include a lysine 113(K113):glutamic acid 195 (E195) salt bridge between actin subunits, thus suggesting a molecular basis for actin catch-slip bonds. This structural mechanism is supported by the suppression of the catch bonds by the single-residue replacements K113 to serine (K113S) and E195 to serine (E195S) on yeast actin. These results demonstrate and provide a structural explanation for actin catchslip bonds, which may provide a mechanoregulatory mechanism to control cell functions by regulating the depolymerization kinetics of force-bearing actin filaments throughout the cytoskeleton.single-molecule force spectroscopy | mechanotransduction | mechanosensing | nemaline myopathy T he actin cytoskeleton, primarily a force-bearing structure, controls the morphology, motility, and adhesion of the cell (1-4). Its core filamentous component, assembled from actin monomers via noncovalent interactions (5), undergoes rapid and controlled polymerization and depolymerization, allowing the dynamic reorganization of the actin cytoskeleton (1, 2).In cells, this dynamic process can be modulated by forces, and this is crucial to mechanosensitivity, mechanotransduction, and cellular adaptations to mechanical stresses (3, 6-8). For example, the assembly, stabilization, and reorganization of the actin stress fiber and the focal adhesion, where actin filaments constantly sustain tension, are induced by externally applied forces (9-12) dependent on myosin-generated contractility (4,8,13,14) and sensitive to substrate rigidity (3, 15, 16). These observations led us to investigate the molecular mechanism by which actin dynamics are regulated by force.The force-regulated kinetics of several molecular interactions important to adhesion and force-bearing functions of cells are governed by catch-slip bonds, in which the interaction is stabilized by tensile force in a low range and destabilized when force exceeds a threshold (17)(18)(19)(20)(21)(22). Various mechanisms, such as the allosteric model based on intramolecular conformational change under forces (23,24) and the sliding-rebinding model based on force-induced formation of new interactions due to intermolecular interface sliding (18,25), have been proposed to provide structural explanations for catch-slip bonds in different molecular interactions.Here we use atomic force microscopy (AFM) force-clamp experiments to determine how force regulates the off-rate of actin depolymerization and to elucidate the structural ...

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“…The stiffness of molecular bonds can be derived from BFP test cycles 29,70 . The ramping phase of each bond event (Fig.…”

Section: Methodsmentioning

confidence: 99%

Dynamic catch of a Thy-1–α5β1+syndecan-4 trimolecular complex

Fiore

Chen

et al. 2014

Nat Commun

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Cancer cell adhesion to the vascular endothelium is a critical step of tumour metastasis. Endothelial surface molecule Thy-1 (CD90) is implicated in the metastatic process through its interactions with integrins and syndecans. However, how Thy-1 supports cell-cell adhesion in a dynamic mechanical environment is not known. Here we show that Thy-1 supports b 1 integrin-and syndecan-4 (Syn4)-mediated contractility-dependent mechanosignalling of melanoma cells. At the single-molecule level, Thy-1 is capable of independently binding a 5 b 1 integrin and syndecan-4 (Syn4) receptors. However, in the presence of both a 5 b 1 and Syn4, the two receptors bind cooperatively to Thy-1, to form a trimolecular complex. This trimolecular complex displays a unique phenomenon we coin 'dynamic catch', characterized by abrupt bond stiffening followed by the formation of catch bonds, where force prolongs the bond lifetime. Thus, we reveal a new class of trimolecular interactions where force strengthens the synergistic binding of two co-receptors and modulates downstream mechanosignalling.

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Measuring Molecular Elasticity by Atomic Force Microscope Cantilever Fluctuations

Cited by 75 publications

References 42 publications

Molecular Stiffness of Selectins

Molecular Stiffness of Selectins

Actin depolymerization under force is governed by lysine 113:glutamic acid 195-mediated catch-slip bonds

Dynamic catch of a Thy-1–α5β1+syndecan-4 trimolecular complex

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